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Sample records for hamster oocyte penetration

  1. Thermostability of sperm nuclei assessed by microinjection into hamster oocytes

    EPA Science Inventory

    Nuclei isolated from spermatozoa of various species (golden hamster, mouse, human, rooster, and the fish tilapia) were heated at 60 degrees-125 degrees C for 20-120 min and then microinjected into hamster oocytes to determine whether they could decondense and develop into pronucl...

  2. DNA (deoxyribonucleic acid) synthesis following microinjection of heterologous sperm and somatic cell nuclei into hamster oocytes

    SciTech Connect

    Naish, S.J.; Perreault, S.D.; Zirkin, B.R.

    1987-01-01

    The authors investigated the ability of the hamster oocyte to initiate DNA synthesis in nuclei differing in basic protein content. DNA synthesis was studied by autoradiography in oocytes that had been incubated in /sup 3/H-thymidine after being parthenogenetically activated by sham microinjection, or microinjected with hamster, mouse, rabbit, or fish sperm nuclei, or hamster hepatocyte nuclei. Within 6 hr of sham or nucleus microinjection, nuclei of each type underwent transformation into pronuclei and synthesized DNA. These results demonstrated that the hamster egg can access and utilize its own and each type of template provided, whether homologous or heterologous. However, pronuclei derived from hamster sperm nuclei were more likely to be synthesizing DNA at 6 hr than pronuclei derived from sperm nuclei of other species. The authors conclude that the mechanisms employed by the hamster oocyte to transform hamster sperm nuclei into pronuclei and to effect DNA synthesis in these nuclei are not specific for the hamster sperm nucleus. Nevertheless, these mechanisms apparently operate more efficiently when the hamster sperm nucleus, rather than a heterologous sperm nucleus, is present.

  3. Changes in the reciprocal position of the first polar body and oocyte chromosome set in golden hamsters.

    PubMed

    Wang, Lingyan; Li, Dexue; Li, Ziyi

    2009-10-01

    The golden hamster is an attractive model organism for studying reproductive physiology, oncology, genetics and virology. In an effort to establish experimental protocols necessary for cloning golden hamsters, we examined changes in the reciprocal position of the FPB (first polar body) and chromosome set of MII (the second meiotic metaphase) oocytes of golden hamsters. Oocytes were collected under three different conditions: (i) oocyte direct recovery from the oviduct of hormonally treated donor; (ii) oocyte recovery from the oviduct of hormonally treated donor followed by 5 h/10 h in vitro culture; and (iii) oocyte recovery from ovaries of hormonally treated donors and in vitro maturation. Then oocyte recovery was performed from the oviduct of hormonally treated donors, followed by 5 h in vitro culture with colchicine and/or CB (cytochalasin B). Denuded oocytes were stained with Hoechst 33342 and propidium iodide and evaluated under a microscope. Our results demonstrate that the change in FPB position in relation to the MII oocyte chromosome set increases with age of in vivo-matured oocytes. Cumulus cells can protect the FPB of in vitro-cultured oocytes from degeneration but do not significantly affect its repositioning, and in vitro-matured oocytes age slower. The colchicine has a stronger effect on cytoplasmic protrusions of golden hamster oocytes when compared with CB. These results define conditions for changes in FPB position relative to the MII oocyte chromosome set. Early ovulated oocytes, in vitro-matured oocytes and oocytes treated with colchicine should improve the effectiveness of the cloning procedure in golden hamsters as an animal model for human diseases. PMID:18980577

  4. Human sperm chromosome analysis after subzonal sperm insemination of hamster oocytes

    SciTech Connect

    Cozzi, J.

    1994-09-01

    Sperm microinjection techniques, subzonal sperm insemination (SUZI) and intracytoplasmic sperm injection (ICSI), have achieved a wide spread clinical application for the treatment of male infertility. To date, only one study has focused on sperm karyotypes after microinjection. Martin et al. reported a very high incidence of abnormal human sperm complements after ICSI into hamster oocytes. In the present study, are reported the first human sperm karyotypes after SUZI of hamster oocytes. Spermatozoa from two control donors were treated by calcium ionophore A23187 and injected under the zona of hamster eggs. The microinjected eggs were then cultured for cytogenetic analysis of the pronuclei. Out of 47 analyzed sperm chromosome metaphases, 5 (10.6%) were abnormal, 4 (8.5%) were hypohaploid and 1 (2.1%) had a structural abnormality. The sex ratio was not significantly different from the expected 1:1 ratio. Rates of chromosomal abnormalities in microinjected spermatozoa were similar to those observed in spermatozoa inseminated with zona free eggs, suggesting that SUZI procedure per se does not increase sperm chromosomal abnormalities.

  5. The application of the zona-free hamster egg test for the prognosis of human in vitro fertilization.

    PubMed

    Foreman, R; Cohen, J; Fehilly, C B; Fishel, S B; Edwards, R G

    1984-09-01

    The zona-free hamster egg test was carried out using spermatozoa from 15 men which consistently failed to fertilize their wives' oocytes in vitro. Spermatozoa from nine of these men fertilized hamster eggs in vitro, indicating that positive results in this assay are an unreliable guide to human in vitro fertilization. Donor spermatozoa were needed to fertilize the wife's oocytes in three of these cases. Nevertheless, the proportion of hamster egg penetration was significantly lower compared with spermatozoa from 15 men who could fertilize their wives' oocytes in vitro. The hamster assay also failed to indicate the establishment of pregnancy.

  6. Down-regulation of membrana granulosa cell gap junctions is correlated with irreversible commitment to resume meiosis in golden Syrian hamster oocytes.

    PubMed

    Racowsky, C; Baldwin, K V; Larabell, C A; DeMarais, A A; Kazilek, C J

    1989-08-01

    One of the currently popular hypotheses for the regulation of meiotic resumption in mammalian oocytes proposes that the preovulatory surge of luteinizing hormone causes down-regulation of follicular gap junctions, which in turn disrupts transfer of a meiotic arrester from the somatic cells into the oocyte. The present study has investigated this hypothesis by examining the integrity of membrana granulosa cell gap junctions during the period of irreversible commitment to maturation of golden Syrian hamster oocytes in vivo. Our results have revealed a significant progressive decrease in the fractional area of cell surface occupied by gap junction membrane with increasing percentage of oocytes irreversibly committed to mature (1.946% and 0.921% fractional gap junction area at 0% and 100% oocytes irreversibly committed to mature, respectively, P less than 0.05). This net loss of membrana granulosa cell gap junctions from the cell surface was accompanied by a significant decrease in density of gap junction particles, whether they were arranged in rectilinear or non-rectilinear packing patterns. Furthermore, the number of gap junction particles per unit area of surface membrane scanned also underwent a significant progressive decrease with increasing percentage of oocytes irreversibly committed to mature. These data with the hamster are consistent with the hypothesis that down-regulation of membrana granulosa cell gap junctions may be of central importance in the regulation of gonadotropic stimulation of meiotic resumption in mammalian oocytes.

  7. Recombinant Hamster Oviductin Is Biologically Active and Exerts Positive Effects on Sperm Functions and Sperm-Oocyte Binding

    PubMed Central

    Yang, Xiaojing; Zhao, Yuewen; Yang, Xiaolong; Kan, Frederick W. K.

    2015-01-01

    Studies carried out in several mammalian species suggest that oviductin, also known as oviduct-specific glycoprotein or OVGP1, plays a key role in sperm capacitation, fertilization, and development of early embryos. In the present study, we used recombinant DNA technology to produce, for the first time, recombinant hamster OVGP1 (rHamOVGP1) in human embryonic kidney 293 (HEK293) cells. rHamOVGP1 secreted in the culture medium was purified by affinity chromatography. The resulting protein migrated as a poly-dispersed band of 160-350 kDa on SDS-PAGE corresponding to the molecular mass of the native HamOVGP1. Subsequent mass spectrometric analysis of the purified rHamOVGP1 confirmed its identity as HamOVGP1. Immunocytochemistry demonstrated binding of rHamOVGP1 to the mid-piece and head of hamster sperm and to the zona pellucida (ZP) of ovarian oocytes. In vitro functional experiments showed that addition of rHamOVGP1 in the capacitation medium further enhanced tyrosine phosphorylation of two sperm proteins of approximately 75 kDa and 83 kDa in a time-dependent manner. After 3 hours of incubation in the presence of rHamOVGP1, a significant increase in acrosome reaction was measured. Pretreatment of either sperm or oocyte with 20 μg/ml of rHamOVGP1 prior to sperm-egg binding assay significantly increased the number of sperm bound to the ZP. Addition of rHamOVGP1 in the medium during sperm-egg binding with either oocyte or sperm pretreated with rHamOVGP1 also saw an increase in the number of sperm bound to ZP. In all experimental conditions, the effect of rHamOVGP1 on sperm-oocyte binding was negated by the addition of monoclonal anti-HamOVGP1 antibody. The successful production and purification of a biologically active rHamOVGP1 will allow further exploration of the function of this glycoprotein in reproductive function. PMID:25849110

  8. Kinematics of hamster sperm during penetration of the cumulus cell matrix.

    PubMed

    Drobnis, E Z; Yudin, A I; Cherr, G N; Katz, D F

    1988-12-01

    During capacitation, mammalian spermatozoa gain the ability to penetrate the cumulus cell matrix (CCM). The role of hyperactivated motility for this capacity is uncertain. In the present study, hamster sperm were observed during penetration and progression through the CCM, and flagellar beat patterns were quantitated by characterization of the underlying flagellar bends. Small numbers of sperm were added to cumulus masses slightly compressed on a slide (150 micron depth), and penetration was videorecorded using interference contrast optics. During penetration of the cumulus surface, sperm did not generate the large flagellar bends and asymmetric beats that are hallmarks of hyperactivation in low viscosity media. Instead, they entered slowly using high-frequency, low-amplitude sinusoidal flagellar motions. Within the CCM, sperm continued to move slowly, and they exhibited three distinct patterns of motility. The first was sinusoidal, produced by alternating, propagated bends: principal bends (PB) moved the head away from the beat midline, with the convex edge of the head leading, and reverse bends (RB) had the opposite curvature. The second pattern was asymmetric and sinusoidal: an extreme RB developed in the distal flagellum, was propagated distally, and was followed by a PB of less curvature. The third motility pattern was a hatchet-like stroke of the sperm head which resulted when an extreme, nonpropagated PB developed slowly in the proximal midpiece, and was released rapidly. In this mode there were no reverse bends, and sperm did not progress. There were subpopulations of capacitating sperm in free-swimming medium which had these same bend types and motility patterns, suggesting that qualitative flagellar movement may not change during CCM penetration. Sperm velocity in the CCM was not strongly correlated with flagellar beat kinematics, suggesting local heterogeneity in cumulus mechanical resistance and/or differences in interaction of the matrix with the

  9. A phosphodiesterase type-5 inhibitor, sildenafil, induces sperm capacitation and penetration into porcine oocytes in a chemically defined medium.

    PubMed

    Ioki, Sumire; Wu, Qing-Shan; Takayama, Osamu; Motohashi, Hideyuki H; Wakai, Takuya; Funahashi, Hiroaki

    2016-02-01

    The present study was undertaken to determine the effect of a phosphodiesterase (PDE) type-5 (cyclic guanosine monophosphate-specific) inhibitor, sildenafil, on capacitation and penetration of boar spermatozoa in a basic chemically defined medium (adenosine- and theophylline-free PGM-tac4). When ejaculated spermatozoa were cultured for 90 minutes in the absence or presence of sildenafil at 2.5 mM, the inhibitor significantly increased the percentage of capacitated/acrosome-reacted spermatozoa, as a result of the chlortetracycline assay. When fresh spermatozoa were co-cultured with oocytes in the presence of sildenafil at a different concentration (0, 2.5, 25, or 250 μM), higher sildenafil concentrations (25 and 250 μM) significantly resulted in higher sperm penetration rates. When oocytes matured in vitro were co-cultured with spermatozoa in the presence of 25 μM sildenafil or 25 mM caffeine benzoate for 8 hours, the incidence of penetrated oocytes did not differ between two groups, whereas the incidence of monospermic oocytes in penetrated one was significantly higher in the presence of sildenafil. Immunocytochemical analysis reported the presence of PDE type-5 on the acrosome region of boar spermatozoa. These results report that regulation of cyclic guanosine monophosphate-specific PDE type-5 by sildenafil somehow can increase the penetrability of boar spermatozoa in vitro.

  10. Encapsulation of sex sorted boar semen: sperm membrane status and oocyte penetration parameters.

    PubMed

    Spinaci, Marcella; Chlapanidas, Theodora; Bucci, Diego; Vallorani, Claudia; Perteghella, Sara; Lucconi, Giulia; Communod, Ricardo; Vigo, Daniele; Galeati, Giovanna; Faustini, Massimo; Torre, Maria Luisa

    2013-03-01

    Although sorted semen is experimentally used for artificial, intrauterine, and intratubal insemination and in vitro fertilization, its commercial application in swine species is still far from a reality. This is because of the low sort rate and the large number of sperm required for routine artificial insemination in the pig, compared with other production animals, and the greater susceptibility of porcine spermatozoa to stress induced by the different sex sorting steps and the postsorting handling protocols. The encapsulation technology could overcome this limitation in vivo, protecting and allowing the slow release of low-dose sorted semen. The aim of this work was to evaluate the impact of the encapsulation process on viability, acrosome integrity, and on the in vitro fertilizing potential of sorted boar semen. Our results indicate that the encapsulation technique does not damage boar sorted semen; in fact, during a 72-hour storage, no differences were observed between liquid-stored sorted semen and encapsulated sorted semen in terms of plasma membrane (39.98 ± 14.38% vs. 44.32 ± 11.72%, respectively) and acrosome integrity (74.32 ± 12.17% vs. 66.07 ± 10.83%, respectively). Encapsulated sorted spermatozoa presented a lower penetration potential than nonencapsulated ones (47.02% vs. 24.57%, respectively, P < 0.0001), and a significant reduction of polyspermic fertilization (60.76% vs. 36.43%, respectively, polyspermic ova/total ova; P < 0.0001). However, no difference (P > 0.05) was observed in terms of total efficiency of fertilization expressed as normospermic oocytes/total oocytes (18.45% vs. 15.43% for sorted diluted and sorted encapsulated semen, respectively). The encapsulation could be an alternative method of storing of pig sex sorted spermatozoa and is potentially a promising technique in order to optimize the use of low dose of sexed spermatozoa in vivo. PMID:23261305

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

    PubMed

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

    2014-05-01

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

  12. Evidence suggesting a role for sperm metalloendoprotease activity in penetration of zona-free hamster eggs by human sperm.

    PubMed

    Díaz-Pérez, E; Thomas, P; Meizel, S

    1988-11-01

    It has been reported that metalloendoprotease (MEP) activity is involved in somatic cell membrane fusion events and in the sea urchin sperm acrosome reaction (AR). MEP activity also has been demonstrated in human and other mammalian sperm. The present study was concerned with investigating whether a human sperm MEP is important in membrane events necessary for sperm egg fusion. Ejaculated human sperm were washed, capacitated in vitro, and preincubated with the competitive MEP inhibitors phosphoramidon (50 microM) or CBZ-L-phenylalanine (1 mM), with 100 microM diethylenetriaminepentaacetic acid (DTPA), a heavy metal chelator, or as controls, with the appropriate solvents. The AR was initiated in vitro with preovulatory human follicular fluid and the sperm washed to dilute inhibitors and then coincubated with zona-free golden hamster eggs (zonae and cumuli removed with trypsin and hyaluronidase, respectively). Eggs were washed after 0.5 h, and the number of sperm remaining bound was counted. After 2.5 h further incubation, the eggs were stained with acetolacmoid or acetoorcein and penetration was assayed by counting the number of decondensed sperm heads per egg (penetration index) and the percent of penetrated eggs. The inhibitor treatments did not decrease the percentage of penetrated eggs (range 80-90%), but a significant reduction in the penetration index was observed. Phosphoramidon reduced the penetration index by 45%, CBZ-L-phenylalanine by 57%, and DTPA by 56%. None of the inhibitors decreased the penetration index or the percentage of penetrated eggs when added directly to suspensions of acrosome-reacted sperm and zona-free eggs at the diluted levels that would have been present after washing inhibitor-treated sperm.(ABSTRACT TRUNCATED AT 250 WORDS)

  13. GLUTATHIONE (GSH) CONCENTRATIONS VARY WITH THE CELL CYCLE IN MATURING HAMSTER OOCYTES, ZYGOTES AND PRE-IMPLANATION EMBRYOS

    EPA Science Inventory

    Abstract
    Glutathione (GSH) is thought to play critical roles in oocyte function including spindle maintenance and provision of reducing power needed to initiate sperm chromatin decondensation. Previous observations that GSH concentrations are higher in mature than immature o...

  14. Molecular cloning and functional expression of a chicken intestinal peptide transporter (cPepT1) in Xenopus oocytes and Chinese hamster ovary cells.

    PubMed

    Chen, Hong; Pan, YuanXiang; Wong, Eric A; Bloomquist, Jeffrey R; Webb, Kenneth E

    2002-03-01

    To study peptide absorption in chickens, an intestinal peptide transporter cDNA (cPepT1) was isolated from a chicken duodenal cDNA library. The cDNA was 2914 bp long and encoded a protein of 714 amino acid residues with an estimated molecular size of 79.3 kDa and an isoelectric point of 7.48. cPepT1 protein is similar60% identical to PepT1 from rabbits, humans, mice, rats and sheep. Sixteen dipeptides, three tripeptides and four tetrapeptides that contained the essential amino acids Met, Lys and(or) Trp were used for functional analysis of cPepT1 in Xenopus oocytes and Chinese hamster ovary cells. For most di- and tripeptides tested, the substrate affinities were in the micromolar range, indicating that cPepT1 has high affinity for these peptides. Lys-Lys and Lys-Trp-Lys were exceptions, with substrate affinities in the millimolar range. Neither free amino acids nor tetrapeptides were transported by cPepT1. Northern blot analysis using a full-length cPepT1 cDNA as the probe demonstrated that cPepT1 is expressed strongly in the duodenum, jejunum and ileum, and at lower levels in kidney and ceca. The present study demonstrated for the first time the presence and functional characteristics of a peptide transport system from an avian species.

  15. Ability of abnormally-shaped human spermatozoa to adhere to and penetrate zona-free hamster eggs: correlation with sperm morphology and postincubation motility.

    PubMed

    Bronson, Richard A; Bronson, Susan K; Oula, Lucila D

    2007-01-01

    A body of evidence indicates that morphologically abnormal human spermatozoa may exhibit impaired ability to fertilize. Yet teratospermia has widely varying etiologies, including associations with varicoceles, following fever, cigarette smoking, and exposure to polychlorinated biphenyls. Abnormalities of sperm shape in mice have also been shown to be associated with autosomal gene mutations. These varying causes of teratospermia could have different molecular consequences reflected in altered sperm function. We studied the ability of morphologically abnormal human sperm to penetrate zona-free hamster eggs as a measure of their ability to undergo an acrosome reaction and gamete membrane fusion. Motile sperm from ejaculates containing 15% normal sperm or less, as judged by World Health Organization (1999) criteria, were recovered by ISolate density centrifugation and capacitated by overnight incubation. Zona-free hamster eggs were inseminated with 1 x 10(6) motile capacitated cells and scored for sperm penetration after 3 hours of coincubation. A significant trend was found between the percent of abnormal spermatozoa within the ejaculate and impaired egg-penetrating ability, reflected in the percent of eggs penetrated, the number of penetrating sperm per egg, and the number of sperm adherent to the oolemma. Because only acrosome-reacted human spermatozoa adhere to the oolemma, these results support the notion that abnormally shaped sperm may exhibit an impaired ability to undergo an acrosome reaction. A correlation was also noted between the loss of motility of sperm following overnight incubation and impairment of their ability to undergo gamete membrane fusion. These results confirm prior findings at the level of the zona pellucida that abnormally shaped sperm exhibit functional abnormalities. However, a wide variation was observed between men in the behavior of such sperm, including occasionally high rates of egg penetration. These observations suggest that

  16. USE OF THE FUNGICIDE CARBENDAZIM AS A MODEL COMPOUND TO DETERMINE THE IMPACT OF ACUTE CHEMICAL EXPOSURE DURING OOCYTE MATURATION AND FERTILIZATION ON PREGNANCY OUTCOME IN THE HAMSTER

    EPA Science Inventory

    Here we use a hamster animal model to identify early pregnancy loss due to an acute chemical exposure to the female during the perifertilization interval. The fungicide carbendazim (methyl 1H-benzimidazole-2-carbamate), a microtubule poison with antimitotic activity, was selected...

  17. Effect of sulfated glycoconjugates on capacitation and the acrosome reaction of bovine and hamster spermatozoa.

    PubMed

    Parrish, J J; Susko-Parrish, J L; Handrow, R R; Ax, R L; First, N L

    1989-12-01

    The effects of sulfated glycoconjugates on the preparation of mammalian sperm for fertilization were investigated. The three sulfated glycoconjugates tested were heparin, dextran sulfate, and the fucose sulfate glycoconjugate (FSG) from the sea urchin egg jelly coat. In vivo, FSG induces the acrosome reaction in sea urchin sperm. Bovine sperm were found to be capacitated by heparin and FSG as judged both by ability of lysophosphatidylcholine (LC) to induce an acrosome reaction and by ability to fertilize bovine oocytes in vitro. The mechanism by which heparin or FSG capacitated bovine sperm appeared similar, since glucose inhibited capacitation by both glycoconjugates. In contrast to effects on bovine sperm, heparin and FSG induced the acrosome reaction in capacitated hamster sperm. When hamster sperm were incubated under noncapacitating conditions, heparin had no effect on capacitation or the acrosome reaction. Three molecular weights (MW) of dextran sulfate (5,000, 8,000, 500,000) were found to capacitate bovine sperm as judged by the ability of LC to induce an acrosome reaction. Whereas bovine sperm incubated with 5,000 or 8,000 MW dextran sulfate fertilized more bovine oocytes than control sperm (P less than 0.05), sperm treated with 500,000 MW dextran sulfate failed to penetrate oocytes. The high-MW dextran sulfate appeared to interact with the zona pellucida and/or sperm to prevent sperm binding. Results suggest that sulfated glycoconjugates may prepare sperm for fertilization across a wide range of species.

  18. Disposition and metabolic profiling of the penetration enhancer Azone. I. In vitro studies: Urinary profiles of hamster, rat, monkey, and man

    SciTech Connect

    Wiechers, J.W.; Drenth, B.F.; Adolfsen, F.A.; Prins, L.; de Zeeuw, R.A. )

    1990-05-01

    Chain-labeled {sup 14}C-Azone was intravenously administered to hamster, monkey, and rat, to compare its metabolic profile with that obtained previously in humans after dermal application. Azone-derived radioactivity was excreted predominantly in the urine for both hamster and monkey, which is similar to the disposition in humans. Metabolic profiling in urine revealed extensive systemic metabolism to occur in all species studied. The main fraction of the metabolites was most polar in man, followed by rat, monkey, and hamster. Traces of the parent compound were detectable only in hamster urine. Although some of the polar major human metabolites were also present in rat urine, the animals were unsuitable for collecting metabolites of Azone observed in humans. In rats, complete cleavage of the dodecyl side chain was ruled out by administering Azone that had been labeled at two distinct positions of the molecule. Additionally, oral administration of Azone to rats resulted in the same metabolic profile as intravenous administration, indicating that gastrointestinal metabolism does not occur or is similar to systemic metabolism.

  19. Porcine oocyte maturation in vitro: role of cAMP and oocyte-secreted factors – A practical approach

    PubMed Central

    APPELTANT, Ruth; SOMFAI, Tamás; MAES, Dominiek; VAN SOOM, Ann; KIKUCHI, Kazuhiro

    2016-01-01

    Polyspermy or the penetration of more than one sperm cell remains a problem during porcine in vitro fertilization (IVF). After in vitro culture of porcine zygotes, only a low percentage of blastocysts develop and their quality is inferior to that of in vivo derived blastocysts. It is unknown whether the cytoplasmic maturation of the oocyte is sufficiently sustained in current in vitro maturation (IVM) procedures. The complex interplay between oocyte and cumulus cells during IVM is a key factor in this process. By focusing on this bidirectional communication, it is possible to control the coordination of cumulus expansion, and nuclear and cytoplasmic maturation during IVM to some extent. Therefore, this review focuses on the regulatory mechanisms between oocytes and cumulus cells to further the development of new in vitro embryo production (IVP) procedures, resulting in less polyspermy and improved oocyte developmental potential. Specifically, we focused on the involvement of cAMP in maturation regulation and function of oocyte-secreted factors (OSFs) in the bidirectional regulatory loop between oocyte and cumulus cells. Our studies suggest that maintaining high cAMP levels in the oocyte during the first half of IVM sustained improved oocyte maturation, resulting in an enhanced response after IVF and cumulus matrix disassembly. Recent research indicated that the addition of OSFs during IVM enhanced the developmental competence of small follicle-derived oocytes, which was stimulated by epidermal growth factor (EGF) via developing EGF-receptor signaling. PMID:27349308

  20. Cryopreservation of starfish oocytes.

    PubMed

    Hamaratoğlu, Fisun; Eroğlu, Ali; Toner, Mehmet; Sadler, Kirsten C

    2005-02-01

    Research from many laboratories over the past several decades indicates that invertebrate oocytes and eggs are extraordinarily difficult to freeze. Since starfish oocytes, eggs, and embryos are an important cell and developmental biology model system, there is great interest to cryopreserve these cells. Previous starfish oocyte cryopreservation studies using slow cooling protocols revealed that these cells are highly sensitive to osmotic stress and form intracellular ice at very high sub-zero temperatures, suggesting that common freezing methodologies may not prove useful. We report here that a short exposure to 1.5 M Me2SO/1 M trehalose in hypotonic salt solution followed by ultra-rapid cooling to cryogenic temperatures allows starfish oocytes to be cryopreserved with the average survival rate of 34% when normalized to control oocytes that were exposed to CPA, but not frozen. On average, 51% of the oocytes in 77% of the batches of frozen oocytes underwent meiotic maturation in response to the starfish maturation hormone, 1-methyladenine. In one experiment, eggs developing from thawed oocytes were capable of being fertilized and two developed into embryos. These data suggests that successful cryopreservation of starfish oocytes is possible, but will need further refinement to increase the numbers of fully competent embryos. PMID:15710368

  1. Cryopreservation of starfish oocytes.

    PubMed

    Hamaratoğlu, Fisun; Eroğlu, Ali; Toner, Mehmet; Sadler, Kirsten C

    2005-02-01

    Research from many laboratories over the past several decades indicates that invertebrate oocytes and eggs are extraordinarily difficult to freeze. Since starfish oocytes, eggs, and embryos are an important cell and developmental biology model system, there is great interest to cryopreserve these cells. Previous starfish oocyte cryopreservation studies using slow cooling protocols revealed that these cells are highly sensitive to osmotic stress and form intracellular ice at very high sub-zero temperatures, suggesting that common freezing methodologies may not prove useful. We report here that a short exposure to 1.5 M Me2SO/1 M trehalose in hypotonic salt solution followed by ultra-rapid cooling to cryogenic temperatures allows starfish oocytes to be cryopreserved with the average survival rate of 34% when normalized to control oocytes that were exposed to CPA, but not frozen. On average, 51% of the oocytes in 77% of the batches of frozen oocytes underwent meiotic maturation in response to the starfish maturation hormone, 1-methyladenine. In one experiment, eggs developing from thawed oocytes were capable of being fertilized and two developed into embryos. These data suggests that successful cryopreservation of starfish oocytes is possible, but will need further refinement to increase the numbers of fully competent embryos.

  2. High-Pressure Freezing Electron Microscopy of Zebrafish Oocytes.

    PubMed

    Kanagaraj, Palsamy; Riedel, Dietmar; Dosch, Roland

    2016-01-01

    Oogenesis is an essential cellular and developmental process to prepare the oocyte for propagation of a species after fertilization. Oocytes of oviparous animals are enormous cells endowed with many, big cellular compartments, which are interconnected through active intracellular transport. The dynamic transport pathways and the big organelles of the oocyte provide the opportunity to study cellular trafficking with outstanding resolution. Hence, oocytes were classically used to investigate cellular compartments. Though many novel regulators of vesicle trafficking have been discovered in yeast, tissue culture cells and invertebrates, recent forward genetic screens in invertebrate and vertebrate oocytes isolated novel control proteins specific to multicellular organisms. Zebrafish is a widely used vertebrate model to study cellular and developmental processes in an entire animal. The transparency of zebrafish embryos allows following cellular events during early development with in vivo imaging. Unfortunately, the active endocytosis of the oocyte also represents a drawback for imaging. The massive amounts of yolk globules prevent the penetration of light-beams and currently make in vivo microscopy a challenge. As a consequence, electron microscopy (EM) still provides the highest resolution to analyze the ultra-structural details of compartments and organelles and the mechanisms controlling many cellular pathways of the oocyte. Among different fixation approaches for EM, High Pressure Freezing (HPF) in combination with freeze substitution significantly improves the samples preservation closest to their natural status. Here, we describe the HPF with freeze substitution embedding method for analyzing cellular processes in zebrafish oocytes using electron microscopy. PMID:27557580

  3. Localization of fucosyl glycoconjugates in human oocytes following insemination for in vitro fertilization.

    PubMed

    Tam, P P; Loong, E P; Chiu, T T

    1990-06-01

    Human oocytes that failed to cleave after insemination were examined for the presence of fucosyl glycoconjugates in the perivitelline space by staining with Ulex europeaus lectin conjugated to fluorescein isothiocynate. Oocytes that formed two or three pronuclei following first insemination always exhibited positive lectin staining similar to that observed with in vitro fertilized mouse oocytes. Among those oocytes that failed to form any pronuclei after the first insemination attempt, only 5% contained lectin positive substances in the perivitelline space. Upon reinsemination, a higher percentage of those oocytes produced lectin-positive materials, although pronuclei were still absent. The appearance of fucosyl glycoconjugates in these oocytes might be the result of the release of cortical granules triggered by sperm penetration or, more likely, due to spontaneous granule discharge in senescent oocytes.

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

    PubMed

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

    2016-04-01

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

  5. Effects of exposure to zearalenone on porcine oocytes and sperm during maturation and fertilization in vitro.

    PubMed

    Sambuu, Rentsenkhand; Takagi, Mitsuhiro; Namula, Zhao; Otoi, Takeshige; Shiga, Satoshi; Rodrigues Dos Santos, Regiane; Fink-Gremmels, Johanna

    2011-09-01

    The influence of acute exposure to zearalenone (ZEN) on porcine oocyte maturation, fertilization or sperm penetration ability during both in vitro maturation and fertilization was evaluated. First, oocytes were cultured in ZEN-containing (0-1000 µg/l) maturation medium and then fertilized. The oocytes maturing in vitro without ZEN were then fertilized in ZEN-containing fertilization medium. The maturation rates of oocytes and penetration ability of sperm decreased significantly in the presence of 1000 µg/l of ZEN. However, neither increases in the rates of degeneration and DNA fragmentation of oocytes nor reductions in normal and polyspermic fertilization were observed. ZEN did not affect the sperm penetration rates; however, 1000 µg/l ZEN had positive effects on normal and polyspermic fertilization rates. Therefore, it can be suggested that an acute exposure of porcine oocytes during maturation and of oocytes and sperm during fertilization to ZEN up to 1000 µg/l may not affect the fertility of the oocytes.

  6. Maternal diabetes and oocyte quality

    PubMed Central

    Wang, Qiang; Moley, Kelle H.

    2016-01-01

    Maternal diabetes has been demonstrated to adversely affect preimplantation embryo development and pregnancy outcomes. Emerging evidence has implicated that these effects are associated with compromised oocyte competence. Several developmental defects during oocyte maturation in diabetic mice have been reported over past decades. Most recently, we further identified the structural, spatial and metabolic dysfunction of mitochondria in oocytes from diabetic mice, suggesting the impaired oocyte quality. These defects in the oocyte may be maternally transmitted to the embryo and then manifested later as developmental abnormalities in preimplantation embryo, congenital malformations, and even metabolic disease in the offspring. In this paper, we briefly review the effects of maternal diabetes on oocyte quality, with a particular emphasis on the mitochondrial dysfunction. The possible connection between dysfunctional oocyte mitochondria and reproductive failure of diabetic females, and the mechanism(s) by which maternal diabetes exerts its effects on the oocyte are also discussed. PMID:20226883

  7. Oocyte Maturation and Development

    PubMed Central

    Verlhac, Marie-Hélène; Terret, Marie-Emilie

    2016-01-01

    Sexual reproduction is essential for many organisms to propagate themselves. It requires the formation of haploid female and male gametes: oocytes and sperms. These specialized cells are generated through meiosis, a particular type of cell division that produces cells with recombined genomes that differ from their parental origin. In this review, we highlight the end process of female meiosis, the divisions per se, and how they can give rise to a functional female gamete preparing itself for the ensuing zygotic development. In particular, we discuss why such an essential process in the propagation of species is so poorly controlled, producing a strong percentage of abnormal female gametes in the end. Eventually, we examine aspects related to the lack of centrosomes in female oocytes, the asymmetry in size of the mammalian oocyte upon division, and in mammals the direct consequences of these long-lived cells in the ovary. PMID:26998245

  8. Cryobiological Characteristics of L-proline in Mammalian Oocyte Cryopreservation

    PubMed Central

    Zhang, Lu; Xue, Xu; Yan, Jie; Yan, Li-Ying; Jin, Xiao-Hu; Zhu, Xiao-Hui; He, Zhi-Zhu; Liu, Jing; Li, Rong; Qiao, Jie

    2016-01-01

    Background: L-proline is a natural, nontoxic cryoprotectant that helps cells and tissues to tolerate freezing in a variety of plants and animals. The use of L-proline in mammalian oocyte cryopreservation is rare. In this study, we explored the cryobiological characteristics of L-proline and evaluated its protective effect in mouse oocyte cryopreservation. Methods: The freezing property of L-proline was detected by Raman spectroscopy and osmometer. Mature oocytes obtained from 8-week-old B6D2F1 mice were vitrified in a solution consisting various concentration of L-proline with a reduced proportion of dimethyl sulfoxide (DMSO) and ethylene glycol (EG), comparing with the control group (15% DMSO and 15% EG without L-proline). The survival rate, 5-methylcytosine (5-mC) expression, fertilization rate, two-cell rate, and blastocyst rate in vitro were assessed by immunofluorescence and in vitro fertilization. Data were analyzed by Chi-square test. Results: L-proline can penetrate the oocyte membrane within 1 min. The osmotic pressure of 2.00 mol/L L-proline mixture is similar to that of the control group. The survival rate of the postthawed oocyte in 2.00 mol/L L-proline combining 7.5% DMSO and 10% EG is significantly higher than that of the control group. There is no difference of 5-mC expression between the L-proline combination groups and control. The fertilization rate, two-cell rate, and blastocyst rate in vitro from oocyte vitrified in 2.00 mol/L L-proline combining 7.5% DMSO and 10% EG solution are similar to that of control. Conclusions: It indicated that an appropriate concentration of L-proline can improve the cryopreservation efficiency of mouse oocytes with low concentrations of DMSO and EG, which may be applicable to human oocyte vitrification. PMID:27503023

  9. The Hamster Cheek Pouch

    PubMed Central

    Klintworth, Gordon K.

    1973-01-01

    To gain insight into factors that might be responsible for the normal avascularity of the cornea and for its vascularization in certain pathologic states, an experimental model was designed in which corneal vascularization could be studied under controlled conditions in hamster cheek pouch chambers. Normal corneal tissue, as well as corneas that had been altered in a variety of ways (eg, boiled, autoclaved, freeze-thawed) were implanted into hamster cheek pouch chambers. The fate of the transplanted tissue was observed at regular intervals by direct visualization within the hamster cheek pouch at various magnifications and by light and electron microscopy. This report reviews observations on more than 300 such experiments. Normal and injured corneal autografts, allografts and xenografts and nonviable (autoclaved, boiled or freeze-thawed) corneas commonly became vascularized in the cheek pouch. When this occurred, a similar morphologic sequence of events preceded and accompanied the growth of blood vessels into the cornea. Vascular invasion was generally preceded by the formation of granulation tissue around the cornea. This was followed by a leukocytic, and frequently a fibroblastic, infiltration of the cornea. When cells did not invade the transplanted cornea, the cornea invariably remained avascular. In the present model, a swollen cornea was not a sufficient stimulus for corneal vascularization. The data suggest that under certain circumstances leukocytes may produce one or more factors which stimulate directional vascular growth. The findings are viewed in terms of current concepts on corneal vascularization. ImagesFig 5Fig 6Fig 7Fig 8Fig 9Fig 10Fig 11Fig 12Fig 1Fig 2Fig 3Fig 4Fig 13Fig 14 PMID:4271966

  10. Gait disturbances in dystrophic hamsters.

    PubMed

    Hampton, Thomas G; Kale, Ajit; Amende, Ivo; Tang, Wenlong; McCue, Scott; Bhagavan, Hemmi N; VanDongen, Case G

    2011-01-01

    The delta-sarcoglycan-deficient hamster is an excellent model to study muscular dystrophy. Gait disturbances, important clinically, have not been described in this animal model. We applied ventral plane videography (DigiGait) to analyze gait in BIO TO-2 dystrophic and BIO F1B control hamsters walking on a transparent treadmill belt. Stride length was ∼13% shorter (P < .05) in TO-2 hamsters at 9 months of age compared to F1B hamsters. Hindlimb propulsion duration, an indicator of muscle strength, was shorter in 9-month-old TO-2 (247 ± 8 ms) compared to F1B hamsters (272 ± 11 ms; P < .05). Braking duration, reflecting generation of ground reaction forces, was delayed in 9-month-old TO-2 (147 ± 6 ms) compared to F1B hamsters (126 ± 8 ms; P < .05). Hindpaw eversion, evidence of muscle weakness, was greater in 9-month-old TO-2 than in F1B hamsters (17.7 ± 1.2° versus 8.7 ± 1.6°; P < .05). Incline and decline walking aggravated gait disturbances in TO-2 hamsters at 3 months of age. Several gait deficits were apparent in TO-2 hamsters at 1 month of age. Quantitative gait analysis demonstrates that dystrophic TO-2 hamsters recapitulate functional aspects of human muscular dystrophy. Early detection of gait abnormalities in a convenient animal model may accelerate the development of therapies for muscular dystrophy.

  11. Penetration equations

    SciTech Connect

    Young, C.W.

    1997-10-01

    In 1967, Sandia National Laboratories published empirical equations to predict penetration into natural earth materials and concrete. Since that time there have been several small changes to the basic equations, and several more additions to the overall technique for predicting penetration into soil, rock, concrete, ice, and frozen soil. The most recent update to the equations was published in 1988, and since that time there have been changes in the equations to better match the expanding data base, especially in concrete penetration. This is a standalone report documenting the latest version of the Young/Sandia penetration equations and related analytical techniques to predict penetration into natural earth materials and concrete. 11 refs., 6 tabs.

  12. Morphological dynamics of cumulus-oocyte complex during oocyte maturation.

    PubMed

    Sato, E

    1998-01-01

    The recent advances on the cytoplasmic regulators of the induction of germinal vesicle break down, maturation and degeneration of oocytes, and glycosaminoglycan composition during cumulus expansion of cumulus-oocyte complexes are discussed. A) Inactive mitogen-activated protein kinases (MAPKs) are present in the oocytes at germinal vesicle (GV) stage, and are activated with germinal vesicle breakdown (GVBD), and remain highly active throughout maturation in porcine oocytes. Inactive MAPKs are localized in the cytoplasm of GV-arrested oocytes and active MAPKs were detected in the GV just before GVBD. B) Cumulus expansion of porcine cumulus-oocyte complexes (COCs) was reduced by oocy tectomy. The profile of total glycosaminoglycan synthesis was attributed to hyaluronic acid rather than chondroitin sulfate in intact COCs and oocytectomy reduced hyaluronic acid synthesis. C) The abnormalities of chromosomes and alpha-tubulin morphology were observed in the oocytes of c-mos deficient mice. MAPK activity of c-mos deficient oocytes did not significantly fluctuate throughout maturation and was clearly lower than that of wild-type oocytes. One of the most drastic abnormalities in c-mos knockout mouse oocytes was their entrance into the interphase instead of second meiosis after first polar body emission. D) Reverse transcriptase/polymerase chain reaction-Southern blot hybridization demonstrated positive expression of Fas in intraovarian mouse oocytes. In contrast, expression of Fas ligand was detected in granulosa cells. These findings were histologically confirmed by in situ hybridization with Fas- and FasL-specific probes. Co-culture of intact and zona-free eggs and granulosa cells demonstrated positive TUNEL staining only zona-free eggs.

  13. Effect of DMSO and DMBA hamster pouch carcinogenesis

    SciTech Connect

    Rivera-Hidalgo, F.; Miller, E.G.; Binnie, W.H.

    1987-01-01

    The penetration of mucosal surfaces by chemical carcinogens is required for tumor induction. The effectiveness of dimethyl sulfoxide (DMSO) as a carrier for carcinogen is controversial. The purpose of this study was to determine what effect DMSO would have on the 9,10-dimethyl- 1,2-benzanthracene (DMBA)-induced carcinogenesis in the hamster cheek pouch. Thirty Syrian golden hamsters were divided into two groups: the control group received a topical application of 0.5% DMBA in mineral oil three times per week for 16 weeks, while the experimental group received a topical application of DMSO previous to each DMBA application. At autopsy, both groups had developed tumors, the tumor ratio of control to experimental was 3.5:1.9 and the average size of tumors was 2.2 to 1.9 mm sq. The results suggest that DMSO interfered with the usual DMBA induction mechanism.

  14. Maturation of Oocytes in Vitro.

    PubMed

    Lonergan, Patrick; Fair, Trudee

    2016-01-01

    Only a fraction of oocytes present in the ovaries at birth are ever ovulated during the lifetime of a female mammal. In vitro maturation (IVM) offers the possibility to exploit what is a largely untapped biological resource. Although IVM is used routinely for the in vitro production of embryos in domestic species, especially cattle, its clinical use in human-assisted reproduction is still evolving. The successful recapitulation in vitro of the events associated with successful oocyte maturation is not always achieved, with the majority of immature oocytes typically failing to develop to the blastocyst stage. Evidence suggests that although culture conditions throughout in vitro embryo production may have a modest influence on the developmental potential of the early embryo, the quality of the oocyte at the start of the process is the key factor determining the proportion of oocytes developing to the blastocyst stage.

  15. Microinjection of follicle-enclosed mouse oocytes

    PubMed Central

    Jaffe, Laurinda A.; Norris, Rachael P.; Freudzon, Marina; Ratzan, William J.; Mehlmann, Lisa M.

    2011-01-01

    Summary The mammalian oocyte develops within a complex of somatic cells known as a follicle, within which signals from the somatic cells regulate the oocyte, and signals from the oocyte regulate the somatic cells. Because isolation of the oocyte from the follicle disrupts these communication pathways, oocyte physiology is best studied within an intact follicle. Here we describe methods for quantitative microinjection of follicle-enclosed mouse oocytes, thus allowing the introduction of signaling molecules as well as optical probes into the oocyte within its physiological environment. PMID:19085139

  16. Microinjection of Follicle-Enclosed Mouse Oocytes

    NASA Astrophysics Data System (ADS)

    Jaffe, Laurinda A.; Norris, Rachael P.; Freudzon, Marina; Ratzan, William J.; Mehlmann, Lisa M.

    The mammalian oocyte develops within a complex of somatic cells known as a follicle, within which signals from the somatic cells regulate the oocyte, and signals from the oocyte regulate the somatic cells. Because isolation of the oocyte from the follicle disrupts these communication pathways, oocyte physiology is best studied within an intact follicle. Here we describe methods for quantitative microinjection of follicle-enclosed mouse oocytes, thus allowing the introduction of signaling molecules as well as optical probes into the oocyte within its physiological environment.

  17. Photoperiod-dependent modulation of anti-Müllerian hormone in female Siberian hamsters, Phodopus sungorus.

    PubMed

    Kabithe, Esther W; Place, Ned J

    2008-03-01

    Fertility and fecundity decline with advancing age in female mammals, but reproductive aging was decelerated in Siberian hamsters (Phodopus sungorus) raised in a short-day (SD) photoperiod. Litter success was significantly improved in older hamsters when reared in SD and the number of primordial follicles was twice that of females held in long days (LD). Because anti-Müllerian hormone (AMH) appears to inhibit the recruitment of primordial follicles in mice, we sought to determine whether the expression patterns of AMH differ in the ovaries and serum of hamsters raised in SD versus LD. Ovaries of SD female hamsters are characterized by a paucity of follicular development beyond the secondary stage and are endowed with an abundance of large eosinophilic cells, which may derive from granulosa cells of oocyte-depleted follicles. In ovaries from 10-week-old SD hamsters, we found that the so-called 'hypertrophied granulosa cells' were immunoreactive for AMH, as were granulosa cells within healthy-appearing primary and secondary follicles. Conversely, ovaries from age-matched LD animals lack the highly eosinophilic cells present in SD ovaries. Therefore, AMH staining in LD was limited to primary and secondary follicles that are comparable in number to those found in SD ovaries. The substantially greater AMH expression in SD ovaries probably reflects the abundance of hypertrophied granulosa cells in SD ovaries and their relative absence in LD ovaries. The modulation of ovarian AMH by day length is a strong mechanistic candidate for the preservation of primordial follicles in female hamsters raised in a SD photoperiod.

  18. Oocyte development, meiosis and aneuploidy.

    PubMed

    MacLennan, Marie; Crichton, James H; Playfoot, Christopher J; Adams, Ian R

    2015-09-01

    Meiosis is one of the defining events in gametogenesis. Male and female germ cells both undergo one round of meiotic cell division during their development in order to reduce the ploidy of the gametes, and thereby maintain the ploidy of the species after fertilisation. However, there are some aspects of meiosis in the female germline, such as the prolonged arrest in dictyate, that appear to predispose oocytes to missegregate their chromosomes and transmit aneuploidies to the next generation. These maternally-derived aneuploidies are particularly problematic in humans where they are major contributors to miscarriage, age-related infertility, and the high incidence of Down's syndrome in human conceptions. This review will discuss how events that occur in foetal oocyte development and during the oocytes' prolonged dictyate arrest can influence meiotic chromosome segregation and the incidence of aneuploidy in adult oocytes.

  19. The rat oocyte synthesises melatonin.

    PubMed

    Sakaguchi, Kenichiro; Itoh, Masanori T; Takahashi, Noriyuki; Tarumi, Wataru; Ishizuka, Bunpei

    2013-01-01

    Melatonin (N-acetyl-5-methoxytryptamine) is an indoleamine originally identified in the pineal gland, where it is synthesised enzymatically from serotonin (5-hydroxytryptamine) by the sequential action of arylalkylamine N-acetyltransferase (AANAT) and acetylserotonin O-methyltransferase (ASMT; also known as hydroxyindole O-methyltransferase). Melatonin directly affects ovarian functions and previous studies have suggested that melatonin is synthesised in the ovary. In the present study, we examined whether AANAT and ASMT are expressed in the adult rat ovary. Reverse transcription-polymerase chain reaction analyses demonstrated that both AANAT and ASMT mRNAs are expressed in the ovary. Western blotting for AANAT protein showed that the ovary, like the pineal gland, contains this enzymatic protein with a molecular mass of 24kDa. Immunohistochemistry revealed that the AANAT protein is localised to the oocyte, corpus luteum and medulla, including mast cells. AANAT protein was found in oocytes at all stages of follicular development, and its levels in oocytes increased progressively throughout follicular development. Furthermore, isolated oocytes metabolised exogenous serotonin to melatonin. These findings demonstrate that melatonin is synthesised from serotonin in oocytes. Melatonin synthesised in the oocyte may be implicated in its own growth or maturation, for example, by acting as a calmodulin antagonist or an antioxidant.

  20. Patterns of intercellular connectivity in the mammalian cumulus-oocyte complex.

    PubMed

    Albertini, D F; Rider, V

    1994-02-01

    Electron and fluorescence microscopic techniques have been used in a complementary fashion to study the patterns of follicle cell-oocyte interactions within cumulus-oocyte-complexes of various mammals. The principal findings are: (1) two distinct types of transzonal processes exist that are distinguishable on the basis of cytoskeletal composition; (2) in some of the species examined (pig, goat, primate), corkscrew-shaped processes rich in tubulin, traverse the zona pellucida and are invaginated into the oocyte cortex; (3) actin-rich processes either ramify as a network at the outer surface of the zona pellucida or penetrate the zona and make contact with the oolemma in a species specific manner. These results are discussed with respect both to the need to employ complementary optical methods in assessing connectivity patterns within COC and to the possible role that extracellular matrix-cell interactions play in the homeostatic control of oocyte growth and maturation.

  1. A morphologic study of unfertilized oocytes and abnormal embryos in human in vitro fertilization.

    PubMed

    Bałakier, H; Casper, R F

    1991-04-01

    The morphology of human, unfertilized oocytes and abnormal embryos cultured in vitro for 48-72 hr was examined in an attempt to learn more about oocyte maturation and reproductive failure in in vitro fertilization (IVF). About 21% of the unfertilized oocytes were totally degenerated. The majority (56%) of the remaining oocytes was arrested at the metaphase II stage. They contained coherent chromosomal plates and had extruded the first polar body with nuclear material. About 13% of oocytes underwent spontaneous activation. In most of these cases the second polar body was retained and many subnuclei or one big nucleus was formed. Five percent of metaphase II oocytes penetrated by sperm were not activated, likely as a result of oocyte immaturity. The developmental ability of abnormal embryos was poor. Several one-cell-stage zygotes were arrested at the pronuclear stage or at mitosis of the first mitotic division. Polyspermic embryos, especially those which contained four or more pronuclei, did not divide or formed uneven, multinucleated blastomeres. However, some triploid and tetraploid embryos often appeared normal morphologically despite their lethal chromosomal abnormalities.

  2. Nanoliter droplet vitrification for oocyte cryopreservation

    PubMed Central

    Zhang, Xiaohui; Khimji, Imran; Shao, Lei; Safaee, Hooman; Desai, Khanjan; Keles, Hasan Onur; Gurkan, Umut Atakan; Kayaalp, Emre; Nureddin, Aida; Anchan, Raymond M; Maas, Richard L; Demirci, Utkan

    2011-01-01

    Aim Oocyte cryopreservation remains largely experimental, with live birth rates of only 2–4% per thawed oocyte. In this study, we present a nanoliter droplet technology for oocyte vitrification. Materials & methods An ejector-based droplet vitrification system was designed to continuously cryopreserve oocytes in nanoliter droplets. Oocyte survival rates, morphologies and parthenogenetic development after each vitrification step were assessed in comparison with fresh oocytes. Results Oocytes were retrieved after cryoprotectant agent loading/unloading, and nanoliter droplet encapsulation showed comparable survival rates to fresh oocytes after 24 h in culture. Also, oocytes recovered after vitrification/thawing showed similar morphologies to those of fresh oocytes. Additionally, the rate of oocyte parthenogenetic activation after nanoliter droplet encapsulation was comparable with that observed for fresh oocytes. This nanoliter droplet technology enables the vitrification of oocytes at higher cooling and warming rates using lower cryoprotectant agent levels (i.e., 1.4 M ethylene glycol, 1.1 M dimethyl sulfoxide and 1 M sucrose), thus making it a potential technology to improve oocyte cryopreservation outcomes. PMID:22188180

  3. Validation of a heterologous fertilization assay and comparison of fertilization rates of equine oocytes using in vitro fertilization, perivitelline, and intracytoplasmic sperm injections.

    PubMed

    Sessions-Bresnahan, D R; Graham, J K; Carnevale, E M

    2014-07-15

    pretreated with sucrose; and (4) ICSI of equine oocytes. Oocytes were examined at 24 hours for cleavage. No equine oocytes cleaved after IVF or PVI. However, ICSI conducted with equine sperm treated with dilauroyl phosphatidylcholine resulted in 85% of the oocytes cleaving. Sperm injected into the PV space of equine oocytes did not appear to enter the ooplasm. This study validated the use of bovine oocytes for equine sperm studies and indicates that failure of equine IVF is more than an inability of equine sperm to penetrate the ZP.

  4. Oocyte aging-induced Neuronatin (NNAT) hypermethylation affects oocyte quality by impairing glucose transport in porcine

    PubMed Central

    Gao, Ying-Ying; Chen, Li; Wang, Tao; Nie, Zheng-Wen; Zhang, Xia; Miao, Yi-Liang

    2016-01-01

    DNA methylation plays important roles in regulating many physiological behaviors; however, few studies were focused on the changes of DNA methylation during oocyte aging. Early studies showed that some imprinted genes’ DNA methylation had been changed in aged mouse oocytes. In this study, we used porcine oocytes to test the hypothesis that oocyte aging would alter DNA methylation pattern of genes and disturb their expression in age oocytes, which affected the developmental potential of oocytes. We compared several different types of genes and found that the expression and DNA methylation of Neuronatin (NNAT) were disturbed in aged oocytes significantly. Additional experiments demonstrated that glucose transport was impaired in aged oocytes and injection of NNAT antibody into fresh oocytes led to the same effects on glucose transport. These results suggest that the expression of NNAT was declined by elevating DNA methylation, which affected oocyte quality by decreasing the ability of glucose transport in aged oocytes. PMID:27782163

  5. Recent progress in reproduction of whale oocytes.

    PubMed

    Zheng, Yue-Liang

    2013-08-01

    Whale oocytes recovered from follicles can be matured in vitro. Whale sperm and mature oocytes can be used for in vitro fertilization (IVF), and IVF embryos have the ability to develop to morula stage. Whale sperm injected into bovine or mouse oocytes can activate the oocytes and form pronucleus. Interspecies somatic cell nuclear transfer embryos have been reconstructed with whale somatic cell nucleus and enucleated bovine or porcine oocytes, and interspecies cloned embryos can develop in vitro. This paper reviews recent progress in maturation, fertilization and development of whale oocytes.

  6. Identification of cigarette smoke components that alter functioning of hamster (Mesocricetus auratus) oviducts in vitro.

    PubMed

    Talbot, P; DiCarlantonio, G; Knoll, M; Gomez, C

    1998-04-01

    Previous studies have shown that solutions of mainstream and sidestream cigarette smoke decrease both ciliary beat frequency and oocyte cumulus complex pick-up rate of hamster oviducts in vitro. The purpose of this study was to identify the component(s) in smoke that produces these effects. Chemicals reported to be ciliotoxic in other systems were tested on hamster infundibula at various concentrations to determine whether a dose-response inhibition of ciliary beat frequency occurred. In addition, the concentration of each test component was measured in mainstream and sidestream smoke solutions (whole, particulate, and gas phase). All test components (acrolein, formaldehyde, phenol, acetaldehyde, and potassium cyanide [KCN]) inhibited ciliary beat frequency in a dose-dependent manner. Inhibition of ciliary beat frequency was at least partially reversible for all test compounds except acrolein. The concentrations of acrolein, formaldehyde, and phenol that were required to inhibit beat frequency were at least 3-50 times higher than their corresponding concentrations in smoke solutions. In contrast, cyanide was present in all smoke solutions at concentrations sufficient to inhibit ciliary beat frequency. Cilia on the outer surface of the infundibulum function in picking up the oocyte cumulus complex. Oocyte cumulus complex pick-up rate was measured in vitro at KCN concentrations shown to inhibit ciliary beat frequency. Pick-up rate was likewise inhibited by KCN and remained significantly depressed after washout of KCN. These data show that cyanide is a potent inhibitor of both ciliary beat frequency and oocyte cumulus complex pick-up rate and that its concentration in smoke solutions is sufficiently high to explain the previously reported inhibition of these oviductal processes.

  7. Oocyte development, meiosis and aneuploidy

    PubMed Central

    MacLennan, Marie; Crichton, James H.; Playfoot, Christopher J.; Adams, Ian R.

    2015-01-01

    Meiosis is one of the defining events in gametogenesis. Male and female germ cells both undergo one round of meiotic cell division during their development in order to reduce the ploidy of the gametes, and thereby maintain the ploidy of the species after fertilisation. However, there are some aspects of meiosis in the female germline, such as the prolonged arrest in dictyate, that appear to predispose oocytes to missegregate their chromosomes and transmit aneuploidies to the next generation. These maternally-derived aneuploidies are particularly problematic in humans where they are major contributors to miscarriage, age-related infertility, and the high incidence of Down's syndrome in human conceptions. This review will discuss how events that occur in foetal oocyte development and during the oocytes’ prolonged dictyate arrest can influence meiotic chromosome segregation and the incidence of aneuploidy in adult oocytes. PMID:26454098

  8. Hamster-tropic sarcomagenic and nonsarcomagenic viruses derived from hamster tumors induced by the Gross pseudotype of Moloney sarcoma virus.

    PubMed

    Kelloff, G; Huebner, R J; Lee, Y K; Toni, R; Gilden, R

    1970-02-01

    Hamster sarcomas induced by the Gross pseudotype of Moloney sarcoma virus yielded a virus sarcomagenic for hamsters but not mice. This virus was able to produce foci on hamster embryo cells, but not on mouse embryo cells. A hamster-tropic nonfocus-forming helper virus was also found in the viral stocks. These hamster-tropic viruses are not immunologically related to the murine viruses in the original inoculum but appear to represent indigenous C-type RNA viruses of the hamster.

  9. Recent Progress in Cryopreservation of Bovine Oocytes

    PubMed Central

    Hochi, Shinichi

    2014-01-01

    Principle of oocyte cryoinjury is first overviewed and then research history of cryopreservation using bovine oocytes is summarized for the last two decades with a few special references to recent progresses. Various types of cryodevices have been developed to accelerate the cooling rate and applied to the oocytes from large domestic species enriched with cytoplasmic lipid droplets. Two recent approaches include the qualitative improvement of IVM oocytes prior to the vitrification and the short-term recovery culture of vitrified-warmed oocytes prior to the subsequent IVF. Supplementation of L-carnitine to IVM medium of bovine oocytes has been reported to reduce the amount of cytoplasmic lipid droplets and improve the cryotolerance of the oocytes, but it is still controversial whether the positive effect of L-carnitine is reproducible. Incidence of multiple aster formation, a possible cause for low developmental potential of vitrified-warmed bovine oocytes, was inhibited by a short-term culture of the postwarm oocytes in the presence of Rho-associated coiled-coil kinase (ROCK) inhibitor. Use of an antioxidant α-tocopherol, instead of the ROCK inhibitor, also supported the revivability of the postwarm bovine oocytes. Further improvements of the vitrification procedure, combined with pre- and postvitrification chemical treatment, would overcome the high sensitivity of bovine oocytes to cryopreservation. PMID:24738063

  10. Cold-induced changes in amphibian oocytes

    SciTech Connect

    Angelier, N.; Moreau, N.A.; N'Da, E.A.; Lautredou, N.F. )

    1989-08-01

    Female Pleurodeles waltl newts (Amphibia, urodele), usually raised at 20 degrees C, were submitted to low temperatures; oocytes responded to this cold stress by drastic changes both in lampbrush chromosome structure and in protein pattern. Preexisting lateral loops of lampbrush chromosomes were reduced in size and number, while cold-induced loops which were tremendously developed, occurred on defined bivalents of the oocyte at constant, reproducible sites. A comparison of protein patterns in control and stressed oocytes showed two main differences: in stressed oocytes, overall protein synthesis was reduced, except for a set of polypeptides, the cold-stress proteins; second, there was a striking inversion of the relative amount of beta- and gamma-actin found in the oocyte nucleus before and after cold stress. Whereas beta-actin was the predominant form in control oocytes, gamma-actin became the major form in stressed oocytes.

  11. Directed Student Inquiry: Modeling in Roborovsky Hamsters

    ERIC Educational Resources Information Center

    Elwess, Nancy L.; Bouchard, Adam

    2007-01-01

    In this inquiry-based activity, Roborovsky hamsters are used to provide students with an opportunity to develop their skills of analysis, inquiry, and design. These hamsters are easy to maintain, yet offer students a means to use conventional techniques and those of their own design to make further observations through measuring, assessing, and…

  12. Effect of dexamethasone on babesiasis in hamsters.

    PubMed

    Eckblad, W P; Stiller, D; Woodard, L F; Kuttler, K L

    1984-09-01

    Three subcutaneous injections of 0.20 mg of dexamethasone/kg of body weight caused a substantial increase in Babesia microti-parasitized RBC of hamsters, indicating that this was a useful method for revealing the presence of latent infections. A relative neutrophilia, lymphocytopenia, and eosinopenia were also seen in the long-term B microti-infected carrier hamsters after 0.20 mg or 0.02 mg of dexamethasone/kg. Noninfected hamsters treated with dexamethasone had a neutrophilic leukocytosis and a transient lymphocytopenia. Spleen to body weight ratios of noninfected hamsters decreased significantly (P less than 0.02) after 4 injections with either dosage level. These ratios did not significantly (P greater than 0.05) decrease in dexamethasone-treated infected hamsters.

  13. 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

  14. How Is Plasminogen/Plasmin System Contributing to Regulate Sperm Entry Into the Oocyte?

    PubMed Central

    Grullón, Luis A.; Gadea, Joaquín; Mondéjar, Irene; Matás, Carmen; Romar, Raquel

    2013-01-01

    Plasminogen is present in the oviduct, on the zona pellucida (ZP) and on oolemma, and reduces the number of sperm penetrating the oocyte during in vitro fertilization in pig and cow. It is unknown how this reduction occurs. We tested whether plasminogen (1) changed the ZP resistance to enzymatic digestion thus making the passage of the spermatozoa across it difficult; (2) reduced the sperm functionality, assessed by sperm viability, motility, spontaneous acrosome reaction and membrane lipid disorder; or (3) affected the sperm–ZP binding before or after sperm–ZP interaction. The mechanism by which plasminogen/plasmin system contributes to regulate sperm entry into the oocyte is not inducing a ZP hardening or a decrease in sperm functionality but detaching more than 50% of sperm bound to the ZP. It is suggested that the fertilizing spermatozoon activates plasminogen into plasmin at the oocyte surface and that plasmin removes additional spermatozoa attached to the ZP. PMID:23420828

  15. G Protein-Coupled Receptor 30 Expression Is Required for Estrogen Stimulation of Primordial Follicle Formation in the Hamster Ovary

    PubMed Central

    Wang, Cheng; Prossnitz, Eric R.; Roy, Shyamal K.

    2008-01-01

    Estradiol-17β (E2) plays an important role in the formation and development of primordial follicles, but the mechanisms remain unclear. G protein-coupled receptor 30 (GPR30) can mediate a rapid and transcription-independent E2 signaling in various cells. The objectives of this study were to examine whether GPR30 was expressed in the neonatal hamster ovary and whether it could mediate estrogen action during the formation of primordial follicles. GPR30 mRNA levels decreased from the 13th day of gestation (E13) through the second day of postnatal (P2) life, followed by steady increases from P3 through P6. Consistent with the changes in mRNA levels, GPR30 protein expression decreased from E13 to P2 followed by a significant increase by P7, the day before the first appearance of primordial follicles in the hamster ovary. GPR30 was expressed both in the oocytes and somatic cells, although the expression in the oocytes was low. GPR30 protein was located primarily in the perinuclear endoplasmic reticulum, which was also the site of E2-BSA-FITC (E2-BSA-fluorescein isothiocyanate) binding. E2 or E2-BSA increased intracellular calcium in neonatal hamster ovary cells in vitro. Exposure to GPR30 small interfering RNA in vitro significantly reduced GPR30 mRNA and protein levels in cultured hamster ovaries, attenuated E-BSA binding to cultured P6 ovarian cells, and markedly suppressed estrogen-stimulated primordial follicle formation. These results suggest that a membrane estrogen receptor, GPR30, is expressed in the ovary during perinatal development and mediates E2 action on primordial follicle formation. PMID:18499747

  16. The capability of reprogramming the male chromatin after fertilization is dependent on the quality of oocyte maturation.

    PubMed

    Gioia, Luisa; Barboni, Barbara; Turriani, Maura; Capacchietti, Giulia; Pistilli, Maria Gabriella; Berardinelli, Paolo; Mattioli, Mauro

    2005-07-01

    The present experiments compared the ability of pig oocytes matured either in vivo or in vitro to structurally reorganize the penetrated sperm chromatin into male pronucleus (PN) and to carry out, in parallel, the epigenetic processes of global chromatin methylation and acetylation, 12-14 h after in vitro fertilization (IVF). In addition, PN distribution of histone deacetylase (HDAC), a major enzyme interfacing DNA methylation and histone acetylation, was investigated. The ability of the oocyte to operate an efficient block to polyspermy was markedly affected by maturation. The monospermic fertilization rate was significantly higher for in vivo than for in vitro matured (IVM) oocytes (P < 0.01) which, furthermore, showed a reduced ability to transform the chromatin of penetrated sperm into male PN (P < 0.01). Indirect immunofluorescence analysis of global DNA methylation, histone acetylation and HDAC distribution (HDAC-1, -2 and -3), carried out in monospermic zygotes that reached the late PN stage, showed that IVM oocytes also had a reduced epigenetic competence. In fact, while in about 80% of in vivo matured and IVF oocytes the male PN underwent a process of active demethylation and showed a condition of histone H4 hyperacetylation, only 40% of IVM/IVF zygotes displayed a similar PN remodelling asymmetry. Oocytes that carried out the first part of maturation in vivo (up to germinal vesicle breakdown; GVBD) and then completed the process in vitro, displayed the same PN asymmetry as oocytes matured entirely in vivo. A crucial role of HDAC in the establishment of PN acetylation asymmetry seems to be confirmed by the use of HDAC inhibitors as well as by the abnormal distribution of the enzyme between the two PN in IVM zygotes. Collectively, these data demonstrated that some pig IVM oocytes fail to acquire full remodelling competence which is independent from their ooplasmic ability to morphologically reorganize the sperm nucleus into PN. PMID:15985629

  17. Laser-assisted in vitro fertilization facilitates fertilization of vitrified-warmed C57BL/6 mouse oocytes with fresh and frozen-thawed spermatozoa, producing live pups.

    PubMed

    Woods, Stephanie E; Qi, Peimin; Rosalia, Elizabeth; Chavarria, Tony; Discua, Allan; Mkandawire, John; Fox, James G; García, Alexis

    2014-01-01

    The utility of cryopreserved mouse gametes for reproduction of transgenic mice depends on development of assisted reproductive technologies, including vitrification of unfertilized mouse oocytes. Due to hardening of the zona pellucida, spermatozoa are often unable to penetrate vitrified-warmed (V-W) oocytes. Laser-assisted in vitro fertilization (LAIVF) facilitates fertilization by allowing easier penetration of spermatozoa through a perforation in the zona. We investigated the efficiency of V-W C57BL/6NTac oocytes drilled by the XYClone laser, compared to fresh oocytes. By using DAP213 for cryoprotection, 83% (1,470/1,762) of vitrified oocytes were recovered after warming and 78% were viable. Four groups were evaluated for two-cell embryo and live offspring efficiency: 1) LAIVF using V-W oocytes, 2) LAIVF using fresh oocytes, 3) conventional IVF using V-W oocytes and 4) conventional IVF using fresh oocytes. First, the groups were tested using fresh C57BL/6NTac spermatozoa (74% motile, 15 million/ml). LAIVF markedly improved the two-cell embryo efficiency using both V-W (76%, 229/298) and fresh oocytes (69%, 135/197), compared to conventional IVF (7%, 12/182; 6%, 14/235, respectively). Then, frozen-thawed C57BL/6NTac spermatozoa (35% motile, 15 million/ml) were used and LAIVF was again found to enhance fertilization efficiency, with two-cell embryo rates of 87% (298/343) using V-W oocytes (P<0.05, compared to fresh spermatozoa), and 73% (195/266) using fresh oocytes. Conventional IVF with frozen-thawed spermatozoa using V-W (6%, 10/168) and fresh (5%, 15/323) oocytes produced few two-cell embryos. Although live offspring efficiency following embryo transfer was greater with conventional IVF (35%, 18/51; LAIVF: 6%, 50/784), advantage was seen with LAIVF in live offspring obtained from total oocytes (5%, 50/1,010; conventional IVF: 2%, 18/908). Our results demonstrated that zona-drilled V-W mouse oocytes can be used for IVF procedures using both fresh and frozen

  18. Laser-Assisted In Vitro Fertilization Facilitates Fertilization of Vitrified-Warmed C57BL/6 Mouse Oocytes with Fresh and Frozen-Thawed Spermatozoa, Producing Live Pups

    PubMed Central

    Woods, Stephanie E.; Qi, Peimin; Rosalia, Elizabeth; Chavarria, Tony; Discua, Allan; Mkandawire, John; Fox, James G.; García, Alexis

    2014-01-01

    The utility of cryopreserved mouse gametes for reproduction of transgenic mice depends on development of assisted reproductive technologies, including vitrification of unfertilized mouse oocytes. Due to hardening of the zona pellucida, spermatozoa are often unable to penetrate vitrified-warmed (V-W) oocytes. Laser-assisted in vitro fertilization (LAIVF) facilitates fertilization by allowing easier penetration of spermatozoa through a perforation in the zona. We investigated the efficiency of V-W C57BL/6NTac oocytes drilled by the XYClone laser, compared to fresh oocytes. By using DAP213 for cryoprotection, 83% (1,470/1,762) of vitrified oocytes were recovered after warming and 78% were viable. Four groups were evaluated for two-cell embryo and live offspring efficiency: 1) LAIVF using V-W oocytes, 2) LAIVF using fresh oocytes, 3) conventional IVF using V-W oocytes and 4) conventional IVF using fresh oocytes. First, the groups were tested using fresh C57BL/6NTac spermatozoa (74% motile, 15 million/ml). LAIVF markedly improved the two-cell embryo efficiency using both V-W (76%, 229/298) and fresh oocytes (69%, 135/197), compared to conventional IVF (7%, 12/182; 6%, 14/235, respectively). Then, frozen-thawed C57BL/6NTac spermatozoa (35% motile, 15 million/ml) were used and LAIVF was again found to enhance fertilization efficiency, with two-cell embryo rates of 87% (298/343) using V-W oocytes (P<0.05, compared to fresh spermatozoa), and 73% (195/266) using fresh oocytes. Conventional IVF with frozen-thawed spermatozoa using V-W (6%, 10/168) and fresh (5%, 15/323) oocytes produced few two-cell embryos. Although live offspring efficiency following embryo transfer was greater with conventional IVF (35%, 18/51; LAIVF: 6%, 50/784), advantage was seen with LAIVF in live offspring obtained from total oocytes (5%, 50/1,010; conventional IVF: 2%, 18/908). Our results demonstrated that zona-drilled V-W mouse oocytes can be used for IVF procedures using both fresh and frozen

  19. Vitrification of buffalo oocytes and embryos.

    PubMed

    Parnpai, Rangsun; Liang, Yuanyuan; Ketudat-Cairns, Mariena; Somfai, Tamas; Nagai, Takashi

    2016-07-01

    During the past decade, vitrification has been acknowledged as an efficient alternative to traditional slow-rate freezing in both human and animal embryology. The buffalo is the major milk and meat producing farm animal in many developing countries. Cryopreservation of buffalo oocytes and embryos is very important in preserving this species for future use. This review discusses the recent buffalo oocytes and embryos vitrification procedures, different types of cryoinjuries, and other factors affecting the vitrification of buffalo oocytes and embryos.

  20. Fourier analysis of mitochondrial distribution in oocytes

    NASA Astrophysics Data System (ADS)

    Hollmann, Joseph L.; Brooks, Dana H.; Newmark, Judith A.; Warner, Carol M.; DiMarzio, Charles A.

    2011-03-01

    This paper describes a novel approach to quantifying mitochondrial patterns which are typically described using the qualitative terms "diffuse" "aggregated" and are potentially key indicators for an oocyte's health and survival potential post-implantation. An oocyte was isolated in a confocal image and a coarse grid was superimposed upon it. The spatial spectrum was calculated and an aggregation factor was generated. A classifier for healthy cells was developed and verified. The aggregation factor showed a clear distinction between the healthy and unhealthy oocytes. The ultimate goal is to screen oocytes for viability preimplantation, thus improving the outcome of in vitro fertilization (IVF) treatments.

  1. Sperm binding capacity of human zona pellucida derived from oocytes obtained from different sources.

    PubMed

    Franken, D R; Kruger, T F; Oehninger, S C; Kaskar, K; Hodgen, G D

    1994-01-01

    The important contributions of sperm-oocyte interaction to infertility diagnostics is well established. Scientists are urged to search for methods to improve the assessment of gamete interaction. Sperm binding and penetration assays have frequented the literature, reporting on various aspects of sperm-oocyte interaction using either microbisected or whole human oocytes during the assay procedure. The objective of the study was to evaluate additional zona pellucida sources which can be used during zona binding studies. Hemizonae were obtained from the following oocytes: 1) experiment 1, prophase I oocytes from post-mortem ovarian tissue from different age groups namely, 7 months, 5 years, 7 years, 12 years and 30 years; 2) experiment 2 used donated immature Prophase I oocytes from the IVF treatment program and 3) experiment 3 evaluated zona binding for hemizonae which were previously used in hemizona assays. Results indicated that, in experiment 1, ovarian age does not have any influence on the zona pellucida's capacity to bind spermatozoa. The mean number of bound sperm among the different age groups did not differ significantly, namely 38.9 +/- 17 (7 months), 31.0 +/- 27 (5 years), 49.3 +/- 21 (7 years), 32.8 +/- 18 (12 years) and 39.5 +/- 17 (30 years). The pooled mean +/- SD binding for all the age groups in experiment 1 was 37.7 +/- 7. Likewise, the mean number of sperm bound (experiment 2) to zonae collected from oocytes using different ovulation induction regimes were 31.1 +/- 20 (unstimulated), 54.4 +/- 12 (HMG/HCG) and 15.3 +/- 9 (HMG alone).(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7825743

  2. Spontaneous endomyometrial neoplasms in aging Chinese hamsters

    SciTech Connect

    Brownstein, D.G.; Brooks, A.L.

    1980-05-01

    Twenty-one endomyometrial neoplasms among 93 nulliparous noninbred Chinese hamsters were evaluated. The median survival time of the 93 females was 1040 days. The median age of hamsters with endomyometrial neoplasms was 1200 days. Neoplasms were classified as carcinomas or malignant mixed muellerian tumors of the endometrium and benign or malignant myometrial neoplasms. There were 13 endometrial adenocarcinomas. Three tumors were mixed adenosquamous carcinomas, which occurred in significantly older Chinese hamsters than did adenocarcinomas. Three malignant mixed muellerian tumors consisted of 2 carcinosarcomas and 1 mixed mesodermal tumor. The 2 myometrial neoplasms were a lelomyoma and a lelomyosarcoma. The classification and relative frequency of these neoplasms were similar to endomyometrial neoplasms of women, which makes Chinese hamsters useful subjects for studies of spontaneous endomyometrial cancers.

  3. Induction of lyme arthritis in LSH hamsters

    SciTech Connect

    Schmitz, J.L.; Schell, R.F.; Hejka, A.; England, D.M.; Konick, L.

    1988-09-01

    In studies of experimental Lyme disease, a major obstacle has been the unavailability of a suitable animal model. We found that irradiated LSH/Ss Lak hamsters developed arthritis after injection of Borrelia burgdorferi in the hind paws. When nonirradiated hamsters were injected in the hind paws with B. burgdorferi, acute transient synovitis was present. A diffuse neutrophilic infiltrate involved the synovia and periarticular structures. The inflammation was associated with edema, hyperemia, and granulation tissue. Numerous spirochetes were seen in the synovial and subsynovial tissues. The histopathologic changes were enhanced in irradiated hamsters. The onset and duration of the induced swelling were dependent on the dose of radiation and the inoculum of spirochetes. Inoculation of irradiated hamsters with Formalin-killed spirochetes or medium in which B. burgdorferi had grown for 7 days failed to induce swelling. This animal model should prove useful for studies of the immune response to B. burgdorferi and the pathogenesis of Lyme arthritis.

  4. Nuclear replacement of in vitro-matured porcine oocytes by a serial centrifugation and fusion method.

    PubMed

    Maedomari, N; Kikuchi, K; Nagai, T; Fahrudin, M; Kaneko, H; Noguchi, J; Nakai, M; Ozawa, M; Somfai, T; Nguyen, L V; Ito, J; Kashiwazaki, N

    2010-08-01

    The objective of the present study was to establish a method for nuclear replacement in metaphase-II (M-II) stage porcine oocytes. Karyoplasts containing M-II chromosomes (K) and cytoplasts without chromosomes (C) were produced from in vitro-matured oocytes by a serial centrifugation method. The oocytes were then reconstructed by fusion of one karyoplast with 1, 2, 3 or 4 cytoplasts (K + 1C, K + 2C, K + 3C and K + 4C, respectively). Reconstructed oocytes, karyoplasts without fusion of any cytoplast (K) and zona-free M-II oocytes (control) were used for experiments. The rates of female pronucleus formation after parthenogenetic activation in all groups of reconstructed oocytes (58.2-77.4%) were not different from those of the K and control groups (58.2% and 66.0%, respectively). In vitro fertilization was carried out to assay the fertilization ability and subsequent embryonic development of the reconstructed oocytes. The cytoplast : karyoplast ratio did not affect the fertilization status (penetration and male pronuclear formation rates) of the oocytes. A significantly high monospermy rate was found in K oocytes (p < 0.05, 61.6%) compared with the other groups (18.2-32.8%). Blastocyst formation rates increased significantly as the number of the cytoplasts fused with karyoplasts increased (p < 0.05, 0.0-15.3%). The blastocyst rate in the K + 4C group (15.3%) was comparable with that of the control (17.8%). Total cell numbers in both the K + 3C and K + 4C groups (16.0 and 15.3 cells, respectively) were comparable with that of the control (26.2 cells). Our results demonstrate that a serial centrifugation and fusion (Centri-Fusion) is an effective method for producing M-II chromosome transferred oocytes with normal fertilization ability and in vitro development. It is suggested that the number of cytoplasts fused with a karyoplast plays a critical role in embryonic development.

  5. Growth and fertilization of porcine fetal oocytes grafted under the renal capsules of nude mice.

    PubMed

    Kaneko, Hiroyuki; Kikuchi, Kazuhiro; Noguchi, Junko

    2016-10-15

    The fetal ovary contains a larger pool of oocytes than the adult ovary, but utilization of the fetal oocytes of large animals has hardly been examined. In this study, we investigated the developmental competence of oocytes grown in host mice harboring ovarian grafts obtained from fetal pigs. Ovarian fragments from fetuses at 55, 70, and 90 days postartificial insemination (dpi) were grafted into ovariectomized nude mice (Crlj:CD1-Foxn1(nu); 55-, 70- and 90-dpi groups, respectively). For comparison, ovarian fragments from 20-day postpartum (dpp) piglets were also grafted (20-dpp group). About 60 days after detection of vaginal opening, the mice were given 62.5 U/mL porcine FSH for 13 days by infusion to enhance their follicular development. In the fetal ovaries before grafting, the percentage of germ cells in primordial follicles (termed primordial oocytes) relative to the total number of germ cells was 0.06% at 55 dpi, 2.4% at 70 dpi, and 7.2% at 90 dpi, but the majority was contained within egg nests. At 20 dpp, primordial oocytes accounted for 91.7% of the total number of germ cells and the rest were mostly in primary follicles. After FSH stimulation of host mice, formation of antral follicles was promoted in the grafts of the 70- and 90-dpi groups as well as the 20-dpp group, but a very small number of antral follicles developed in the 55-dpi group consistent with the lowest (P < 0.05) levels of circulating inhibin in that group. The mean number of full-sized oocytes with meiotic competence recovered per mouse was 6.0 in the 70-dpi, 18.0 in the 90-dpi, and 21.2 in the 20-dpp groups, whereas virtually no oocytes were recovered from mice in the 55-dpi group. Moreover, the mature oocytes in the 70- and 90-dpi groups were fertilized in vitro, as shown by formation of male and female pronuclei, but the percentage of oocytes penetrated by sperm was low in the 70- (49%) and 90-dpi (29%) groups as compared with the 20-dpp group (88%). These results clearly

  6. SV40 lymphomagenesis in Syrian golden hamsters

    PubMed Central

    McNees, Adrienne L.; Vilchez, Regis A.; Heard, Tiffany C.; Sroller, Vojtech; Wong, Connie; Herron, Alan J.; Hamilton, Mary J.; Davis, William C.; Butel, Janet S.

    2013-01-01

    Simian virus 40 (SV40) isolates differ in oncogenic potential in Syrian golden hamsters following intraperitoneal inoculation. Here we describe the effect of intravenous exposure on tumor induction by SV40. Strains SVCPC (simple regulatory region) and VA45-54(2E) (complex regulatory region) were highly oncogenic following intravenous inoculation, producing a spectrum of tumor types. Three lymphoma cell lines were established; all expressed SV40 T-antigen, were immortalized for growth in culture, and were tumorigenic following transplantation in vivo. New monoclonal antibodies directed against hamster lymphocyte surface antigens are described. The cell lines expressed MHC class II and macrophage markers and were highly phagocytic, indicating a histiocytic origin. Many hamsters that remained tumor-free developed SV40 T-antigen antibodies, suggesting that viral replication occurred. This study shows that route of exposure influences the pathogenesis of SV40-mediated carcinogenesis, that SV40 strain VA45-54(2E) is lymphomagenic in hamsters, that hamster lymphoid cells of histiocytic origin can be transformed in vivo and established in culture, and that reagents to hamster leukocyte differentiation molecules are now available. PMID:19038412

  7. 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

  8. Taurine protects hamster bronchioles from acute NO2-induced alterations. A histologic, ultrastructural, and freeze-fracture study.

    PubMed

    Gordon, R E; Shaked, A A; Solano, D F

    1986-12-01

    In this study the authors describe the use of dietary taurine to protect hamster lung epithelium from acute nitrogen dioxide (NO2) injury. The conclusions were based on histologic, ultrastructural, and freeze-fracture analyses. Hamsters were pretreated for 14 days with 0.5% taurine in their drinking water. They were then exposed to either 7 or 30 ppm NO2 for 24 hours. The lungs from animals of these experimental groups were compared with those from hamsters treated with only NO2, and those given only taurine and with untreated controls. After treatment, hamsters were anesthetized and perfusion-fixed through the right side of the heart with a solution containing 1% glutaraldehyde, 4% paraformaldehyde, and 0.2 M cacodylate. The trachea and lungs were removed en bloc and stored overnight in cacodylate buffer at 4 C. Terminal and respiratory bronchioles, including alveolar ducts and peribronchiolar alveoli, were dissected from each lobe and processed for embedding in Epon and freeze-fracture replication. Light and transmission electron microscopy revealed the typical inflammatory cell infiltrate in the bronchiolar and alveolar duct regions in the lungs of hamsters exposed to NO2. The bronchiolar epithelium appeared flattened because of loss and breakage of cilia on ciliated cells and apical protrusions of Clara cells. Clara-cell secretory granules were reduced or absent. Freeze-fracture replicas of tight junctions of bronchiolar epithelium analyzed by morphometric techniques demonstrated a reduction and fragmentation of fibrils. Only animals exposed to 30 ppm NO2 exhibited physiologic intercellular penetration of horseradish peroxidase. Hamsters pretreated with taurine and then exposed to NO2 showed none of these alterations. They exhibited the same morphologic features as the untreated controls and the hamsters treated only with taurine. On the basis of this evidence, it is suggested that prophylactic dietary taurine can prevent acute NO2-induced morphologic lung

  9. Control of Oocyte Reawakening by Kit.

    PubMed

    Saatcioglu, Hatice Duygu; Cuevas, Ileana; Castrillon, Diego H

    2016-08-01

    In mammals, females are born with finite numbers of oocytes stockpiled as primordial follicles. Oocytes are "reawakened" via an ovarian-intrinsic process that initiates their growth. The forkhead transcription factor Foxo3 controls reawakening downstream of PI3K-AKT signaling. However, the identity of the presumptive upstream cell surface receptor controlling the PI3K-AKT-Foxo3 axis has been questioned. Here we show that the receptor tyrosine kinase Kit controls reawakening. Oocyte-specific expression of a novel constitutively-active KitD818V allele resulted in female sterility and ovarian failure due to global oocyte reawakening. To confirm this result, we engineered a novel loss-of-function allele, KitL. Kit inactivation within oocytes also led to premature ovarian failure, albeit via a contrasting phenotype. Despite normal initial complements of primordial follicles, oocytes remained dormant with arrested oocyte maturation. Foxo3 protein localization in the nucleus versus cytoplasm explained both mutant phenotypes. These genetic studies provide formal genetic proof that Kit controls oocyte reawakening, focusing future investigations into the causes of primary ovarian insufficiency and ovarian aging. PMID:27500836

  10. Mammalian oocyte development: checkpoints for competence.

    PubMed

    Fair, Trudee

    2010-01-01

    During the lifespan of the female, biochemical changes occur in the ovarian environment. These changes are brought about by numerous endogenous and exogenous factors, including husbandry practices, production demands and disease, and can have a profound effect on ovarian oocyte quality and subsequent embryo development. Despite many investigations, there is no consensus regarding the time or period of follicular oocyte development that is particularly sensitive to insult. Here, the key molecular and morphological events that occur during oocyte and follicle growth are reviewed, with a specific focus on identifying critical checkpoints in oocyte development. The secondary follicle stage appears to be a key phase in follicular oocyte development because major events such as activation of the oocyte transcriptome, sequestration of the zona pellucida, establishment of bidirectional communication between the granulosa cells and the oocyte and cortical granule synthesis occur during this period of development. Several months later, the periovulatory period is also characterised by the occurrence of critical events, including appropriate degradation or polyadenylation of mRNA transcripts, resumption of meiosis, spindle formation, chromosome alignment and segregation, and so should also be considered as a potential checkpoint of oocyte development.

  11. Control of Oocyte Reawakening by Kit

    PubMed Central

    Castrillon, Diego H.

    2016-01-01

    In mammals, females are born with finite numbers of oocytes stockpiled as primordial follicles. Oocytes are “reawakened” via an ovarian-intrinsic process that initiates their growth. The forkhead transcription factor Foxo3 controls reawakening downstream of PI3K-AKT signaling. However, the identity of the presumptive upstream cell surface receptor controlling the PI3K-AKT-Foxo3 axis has been questioned. Here we show that the receptor tyrosine kinase Kit controls reawakening. Oocyte-specific expression of a novel constitutively-active KitD818V allele resulted in female sterility and ovarian failure due to global oocyte reawakening. To confirm this result, we engineered a novel loss-of-function allele, KitL. Kit inactivation within oocytes also led to premature ovarian failure, albeit via a contrasting phenotype. Despite normal initial complements of primordial follicles, oocytes remained dormant with arrested oocyte maturation. Foxo3 protein localization in the nucleus versus cytoplasm explained both mutant phenotypes. These genetic studies provide formal genetic proof that Kit controls oocyte reawakening, focusing future investigations into the causes of primary ovarian insufficiency and ovarian aging. PMID:27500836

  12. Relationship between the number of cells surrounding oocytes and energy states of oocytes.

    PubMed

    Munakata, Yasuhisa; Ichinose, Tomoya; Ogawa, Kaori; Itami, Nobuhiko; Tasaki, Hidetaka; Shirasuna, Koumei; Kuwayama, Takehito; Iwata, Hisataka

    2016-10-15

    Lipid content, ATP content, and histone acetylation are thought to reflect the energy state of cells. In addition, the energy state closely associates with growth and developmental ability of oocytes. Oocyte growth is accompanied by active proliferation of the surrounding granulosa cells (GCs), and GCs play a key role in the provision of energy substrates to the oocytes. In the present study, we first examined the relationship among the average number of GCs per follicle, the average number of cumulus cells (CCs) per oocyte, and the average lipid content in oocytes that developed in vivo within individual donor gilts. Second, we validated the relationship between the number of cells surrounding oocytes and the energy states of oocytes by using an IVC system of oocyte granulosa cell complexes (OGCs) derived from early antral follicles. We collected cumulus cells and oocyte complexes (COCs) from antral follicles (3-5 mm in diameter) and found that average lipid content in oocytes significantly correlated with the average number of both GCs/follicle and CCs/oocyte (P < 0.05). In the next series of experiments, we collected OGCs from early antral follicles (0.5-0.7 mm in diameter), and cultured them for 14 days, and then determined the cell number of OGCs, as well as the lipid content, ATP content, and acetylation level of H4K12 in oocytes grown in vitro. In addition, glucose consumption by OGCs was calculated from the sample media collected at Days 13 and 14. The lipid content of oocytes grown in vitro, significantly correlated with the number of cells surrounding the oocytes (P < 0.01) and with the level of glucose consumption by OGCs (P < 0.01). In addition, both ATP content and H4K12 acetylation levels of oocytes grown in vitro significantly correlated with the number of cells surrounding the oocytes (P < 0.05) and glucose consumption by OGCs (P < 0.05). In conclusion, the lipid content of oocytes depends on the number of cells surrounding the

  13. Oocyte donation: particular technical and ethical aspects.

    PubMed

    Englert, Y; Govaerts, I

    1998-05-01

    This paper analyses the reasons that oocyte and sperm donation are experienced very differently by couples, despite their apparent similarity, and stresses the impact of the difficulties on donor recruitment in all oocyte donation programmes. The various types of donors (occasional, relational, in-vitro fertilization patient and professional) are described together with their motivations, resistance, advantages and disadvantages. The contradictory consequences with free or paid donation, the particular risks of oocyte donation (in comparison with sperm donation) both for the donor and for the recipient are highlighted. The problem of maintaining anonymity is then analysed in ethical terms but also in terms of technical efficacy. A strategy is described which, due to the decision of retaining anonymity, authorizes the sharing of oocytes between recipients. This has as a consequence, an increase in treatment efficacy by avoiding wastage of oocytes offered as a donation. PMID:9665329

  14. Sirtuin Inhibition Adversely Affects Porcine Oocyte Meiosis

    PubMed Central

    Zhang, Liang; Ma, Rujun; Hu, Jin; Ding, Xiaolin; Xu, Yinxue

    2015-01-01

    Sirtuins have been implicated in diverse biological processes, including oxidative stress, energy metabolism, cell migration, and aging. Here, we employed Sirtuin inhibitors, nicotinamide (NAM) and Sirtinol, to investigate their effects on porcine oocyte maturation respectively. The rate of polar body extrusion in porcine oocytes decreased after treatment with NAM and Sirtinol, accompanied with the failure of cumulus cell expansion. We further found that NAM and Sirtinol significantly disrupted oocyte polarity, and inhibited the formation of actin cap and cortical granule-free domain (CGFD). Moreover, the abnormal spindles and misaligned chromosomes were readily detected during porcine oocyte maturation after treatment with NAM and Sirtinol. Together, these results suggest that Sirtuins are involved in cortical polarity and spindle organization in porcine oocytes. PMID:26176547

  15. Precision-cut hamster liver slices as an ex vivo model to study amoebic liver abscess.

    PubMed

    Carranza-Rosales, Pilar; Santiago-Mauricio, María Guadalupe; Guzmán-Delgado, Nancy Elena; Vargas-Villarreal, Javier; Lozano-Garza, Gerardo; Ventura-Juárez, Javier; Balderas-Rentería, Isaías; Morán-Martínez, Javier; Gandolfi, A Jay

    2010-10-01

    Entamoeba histolytica is the etiological agent of amoebiasis, the second cause of global morbidity and mortality due to parasitic diseases in humans. In approximately 1% of the cases, amoebas penetrate the intestinal mucosa and spread to other organs, producing extra-intestinal lesions, among which amoebic liver abscess (ALA) is the most common. To study ALA, in vivo and in vitro models are used. However, animal models may pose ethical issues, and are time-consuming and costly; and cell cultures represent isolated cellular lineages. The present study reports the infection of precision-cut hamster liver slices with Entamoeba histolytica trophozoites. The infection time-course, including tissue damage, parallels findings previously reported in the animal model. At the same time amoebic virulence factors were detected in the infected slices. This new model to study ALA is simple and reproducible, and employs less than 1/3 of the hamsters required for in vivo analyses.

  16. Precision-cut hamster liver slices as an ex vivo model to study amoebic liver abscess.

    PubMed

    Carranza-Rosales, Pilar; Santiago-Mauricio, María Guadalupe; Guzmán-Delgado, Nancy Elena; Vargas-Villarreal, Javier; Lozano-Garza, Gerardo; Ventura-Juárez, Javier; Balderas-Rentería, Isaías; Morán-Martínez, Javier; Gandolfi, A Jay

    2010-10-01

    Entamoeba histolytica is the etiological agent of amoebiasis, the second cause of global morbidity and mortality due to parasitic diseases in humans. In approximately 1% of the cases, amoebas penetrate the intestinal mucosa and spread to other organs, producing extra-intestinal lesions, among which amoebic liver abscess (ALA) is the most common. To study ALA, in vivo and in vitro models are used. However, animal models may pose ethical issues, and are time-consuming and costly; and cell cultures represent isolated cellular lineages. The present study reports the infection of precision-cut hamster liver slices with Entamoeba histolytica trophozoites. The infection time-course, including tissue damage, parallels findings previously reported in the animal model. At the same time amoebic virulence factors were detected in the infected slices. This new model to study ALA is simple and reproducible, and employs less than 1/3 of the hamsters required for in vivo analyses. PMID:20412797

  17. Oocyte cryopreservation: advances and drawbacks.

    PubMed

    Dovey, S

    2012-12-01

    The field of oocyte cryopreservation (OC) had advanced dramatically since the first reported birth from cryopreserved oocytes in 1986, with a significant increase in pregnancy rates described over the past 5 years due to improvements in vitrification technology, a cryopreservation method which virtually means to achieve a "glass-like" state through avoidance of ice formation. The potential clinical benefits of achieving efficient OC protocols have long been recognized. Specifically, OC can be offered to women who face fertility-threatening situations such as therapy for cancer or rheumatologic disease, premature ovarian insufficiency, or need for ovarian surgery as a measure to preserve fertility. Moreover, many women who plan to delay childbearing are interested in pursuing OC in order to protect against age-related fertility decline. For infertility practices, efficient OC technology stands to dramatically streamline donor egg programs, and is a helpful adjuvant in situations where sperm is unexpectedly unavailable at the time of egg retrieval and for couples who do not wish to cryopreserve supernumerary embryos created from in vitro fertilization for moral / ethical reasons. This review will describe the history of OC technology over the past three decades, discuss clinical circumstances for its implementation, and address areas where more research is needed. Given the remarkable improvements in pregnancy rates witnessed over the past five years, OC is certain to play a much larger role in reproductive medicine over the coming decades. PMID:23232533

  18. Polypeptide heterogeneity of hamster and calf fibronectins.

    PubMed Central

    Pena, S D; Mills, G; Hughes, R C; Aplin, J D

    1980-01-01

    The adhesive glycoprotein fibronectin has been isolated from fresh hamster plasma by affinity chromatography on gelatin coupled to Sepharose beads by the method of Engvall & Ruoslahti [Int. J. Cancer (1979) 20, 1-5]. Polyacrylamide-gel electrophoresis of material heated in sodium dodecyl sulphate and 2-mercaptoethanol shows two prominent polypeptide subunits of approx. mol.wts. 215 000 and 200 000, with variable amounts of lower-molecular-weight fragments. The unexpected polypeptide heterogeneity of different preparations of hamster fibronectins and bovine serum fibronectin is shown to be partly an artefact and is generated during isolation and storage of purified fibronectin. Treatment of each hamster fibronectin subunit or a smaller fragment of approx. mol.wt. 140 000 with thermolysin or trypsin after radioiodination produces similar patterns of tyrpsine-containing peptides, indicating similar primary amino-acid sequences. Antibodies raised against the major subunits of hamster plasma fibronectin were coupled to Sepharose beads and used in conjunction with gelatin affinity chromatography to isolate fibronectins extracted with urea from baby-hamster kidney (BHK) cells and present in the long-term culture medium of these cells. The cell and medium fibronectins are similar to hamster plasma fibronectin in amino-acid and carbohydrate composition and also produce very similar peptide 'maps'. We conclude that the various forms of hamster fibronectins are structurally analogous in agreement with indistinguishable biological properties in mediating the substance adhesion of BKH cells [Pena & Hughes (1978) Cell Biol. Int. Rep. 3, 339-344]. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 6. Fig. 7. PMID:7458916

  19. Fluorescent penetrant inspection

    NASA Technical Reports Server (NTRS)

    Sastri, Sankar

    1990-01-01

    The purpose of this experiment is to familiarize the student with fluorescent penetrant inspection and to relate it to classification of various defects. The penetrant method of nondestructive testing is a method for finding discontinuities open to the surface in solids and essentially nonporous bodies. The method employs a penetrating liquid which is applied over the surface and enters the discontinuity or crack. After the excess of penetrant has been cleaned from the surface, the penetrant which exudes or is drawn back out of the crack indicates the presence and location of a discontinuity. The experimental procedure is described.

  20. FAA fluorescent penetrant activities

    SciTech Connect

    Moore, D.G.; Larson, B.F.

    1997-11-01

    The Federal Aviation Administration`s Airworthiness Assurance NDI Validation Center (AANC) and the Center for Aviation Systems Reliability (CASR) are currently working to develop a liquid penetrant inspection (LPI) system evaluation capability that will support the needs of the penetrant manufacturers, commercial airline industry and the FAA. The main focus of this facility is to support the evaluation of penetrant inspection materials, penetrant systems and to apply resources to support industry needs. This paper discusses efforts to create such a facility and an initial project to produce fatigue crack specimens for evaluation of Type 1 penetrant sensitivities.

  1. The susceptibility of the hamster to mouse encephalomyelitis virus.

    PubMed

    DEAN, D J; DALLDORF, G

    1948-12-01

    The OT strain of mouse encephalomyelitis virus induces an inapparent infection in suckling hamsters associated with lesions of the central nervous system and skeletal muscles. The virus increases in pathogenicity after alternating mouse-hamster transfers and then induces both paralysis and encephalitis. Pathogenicity is lost through serial hamster passages but is restored by a single mouse transfer.

  2. Subcutaneous Angiolipoma of Abdomen in a Golden Hamster (Mesocrietus auratus).

    PubMed

    Kondo, H; Sato, T; Shibuya, H; Onuma, M

    2005-10-01

    This is a single case report of an angiolipoma located in the subcutis of a 2-year-old golden hamster. The histological appearance of the tumour resembled that described in other species. The hamster died 1 month following removal of tumour and a necropsy was not performed. This is apparently the first recorded case of angiolipoma in a hamster.

  3. Ca2+ signaling differentiation during oocyte maturation.

    PubMed

    Machaca, Khaled

    2007-11-01

    Oocyte maturation is an essential cellular differentiation pathway that prepares the egg for activation at fertilization leading to the initiation of embryogenesis. An integral attribute of oocyte maturation is the remodeling of Ca2+ signaling pathways endowing the egg with the capacity to produce a specialized Ca2+ transient at fertilization that is necessary and sufficient for egg activation. Consequently, mechanistic elucidation of Ca2+ signaling differentiation during oocyte maturation is fundamental to our understanding of egg activation, and offers a glimpse into Ca2+ signaling regulation during the cell cycle.

  4. Histopathology of Lyme arthritis in LSH hamsters

    SciTech Connect

    Hejka, A.; Schmitz, J.L.; England, D.M.; Callister, S.M.; Schell, R.F.

    1989-05-01

    The authors studied the histopathologic evolution of arthritis in nonirradiated and irradiated hamsters infected with Borrelia burgdorferi. Nonirradiated hamsters injected in the hind paws with B. burgdorferi developed an acute inflammatory reaction involving the synovium, periarticular soft tissues, and dermis. This acute inflammatory reaction was short-lived and was replaced by a mild chronic synovitis as the number of detectable spirochetes in the synovium, periarticular soft tissues, and perineurovascular areas diminished. Exposing hamsters to radiation before inoculation with B. burgdorferi exacerbated and prolonged the acute inflammatory phase. Spirochetes also persisted longer in the periarticular soft tissues. A major histopathologic finding was destructive and erosive bone changes of the hind paws, which resulted in deformation of the joints. These studies should be helpful in defining the immune mechanism participating in the onset, progression, and resolution of Lyme arthritis.

  5. A neuroethological approach to hamster vision.

    PubMed

    Finlay, B L; Sengelaub, D R; Berg, A T; Cairns, S J

    1980-12-01

    The contributions of the midbrain optic tectum to visuomotor behaviors likely to be important to hamsters in the wild were studied, including aperture detection, insect catching, and barrier avoidance. Following tectal undercuts, hamsters ceased to make direct approaches to apertures in the posterior 180 degrees of the visual field; this appeared to be mediated by a loss of exploratory or scanning head movements. Reorientation to and pursuit of crickets jumping out of grasp into the visual periphery was impaired, though initial approach to them was not. Barrier avoidance was unaffected by tectal undercuts. This pattern is similar to the contribution of the frog and toad optic tectum to analogous visuomotor tasks. The contribution of the tectum to searching and scanning in the hamster is an extension of the basic orienting capabilities dependent on optic tectum in anurans.

  6. Decreased adult neurogenesis in hibernating Syrian hamster.

    PubMed

    León-Espinosa, Gonzalo; García, Esther; Gómez-Pinedo, Ulises; Hernández, Félix; DeFelipe, Javier; Ávila, Jesús

    2016-10-01

    Generation of new neurons from adult neural stem cells occurs in the dentate gyrus (DG) of the hippocampus and the lateral walls of the lateral ventricles. In this article, we study the neurogenesis that takes place during the hibernation of the Syrian hamster (Mesocricetus auratus). Using a variety of standard neurogenesis markers and 5-bromo-2-deoxyuridine (BrdU) incorporation, we describe a preferential decrease in the proliferation of newborn neurons in the subventricular zone (SVZ) of the hibernating hamsters (torpor) rather than in the hippocampus. Furthermore, we demonstrate that the proliferative capacity is recovered after 3-4days of torpor when arousal is triggered under natural conditions (i.e., not artificially provoked). In addition, we show that tau3R, a tau isoform with three microtubule-binding domains, is a suitable marker to study neurogenesis both in the SVZ and subgranular zone (SGZ) of the Syrian hamster brain. PMID:27436535

  7. Expression of FSH receptor in the hamster ovary during perinatal development

    PubMed Central

    Chakraborty, Prabuddha; Roy, Shyamal K.

    2014-01-01

    FSH plays an important role in ovarian follicular development, and it functions via the G-protein coupled FSH receptor. The objectives of the present study were to determine if full-length FSHR mRNA and corresponding protein were expressed in fetal through postnatal hamster ovaries to explain the FSH-induced primordial follicle formation, and if FSH or estrogen (E) would affect the expression. A full-length and two alternately spliced FSHR transcripts were expressed from E14 through P20. The level of the full-length FSHR mRNA increased markedly through P7 before stabilizing at a lower level with the formation and activation of primordial follicles. A predicted 87kDa FSHR protein band was detected in fetal through P4 ovaries, but additional bands appeared as ovary developed. FSHR immunosignal was present in undifferentiated somatic cells and oocytes in early postnatal ovaries, but was granulosa cells specific after follicles formed. Both eCG and E significantly up-regulated full-length FSHR mRNA levels. Therefore, FSHR is expressed in the hamster ovary from the fetal life to account for FSH-induced primordial follicle formation and cAMP production. Further, FSH or E regulates the receptor expression. PMID:25462586

  8. Follicular Oocytes Better Support Development in Rabbit Cloning Than Oviductal Oocytes

    PubMed Central

    Sung, Li-Ying; Chen, Chien-Hong; Xu, Jie; Lin, Tzu-An; Su, Hwa-Yun; Chang, Wei-Fang; Liu, Chia-Chia; Sung, Yun-Shao; Cheng, Winston T.K.; Zhang, Jifeng; Tian, X. Cindy; Ju, Jyh-Cherng; Chen, Y. Eugene

    2011-01-01

    Abstract This study was conducted to determine the effect of rabbit oocytes collected from ovaries or oviducts on the developmental potential of nuclear transplant embryos. Donor nuclei were obtained from adult skin fibroblasts, cumulus cells, and embryonic blastomeres. Rabbit oocytes were flushed from the oviducts (oviductal oocytes) or aspirated from the ovaries (follicular oocytes) of superovulated does at 10, 11, or 12 h post-hCG injection. The majority of collected oocytes were still attached to the sites of ovulation on the ovaries. We found that follicular oocytes had a significantly higher rate of fusion with nuclear donor cells than oviductal oocytes. There was no difference in the cleavage rate between follicular and oviductal groups, but morula and blastocyst development was significantly higher in the follicular group than in the oviductal group. Two live clones were produced in follicular group using blastomere and cumulus nuclear donors, whereas one live clone was produced in the oviductal group using a cumulus nuclear donor. These results demonstrate that cloned rabbit embryos derived from follicular oocytes have better developmental competence than those derived from oviductal oocytes. PMID:22029417

  9. The effect of friction and impact angle on the spermatozoa-oocyte local contact dynamics.

    PubMed

    Hedrih, Andjelka; Banić, Milan

    2016-03-21

    Although a large proportion of biomolecules involved in spermatozoa-oocyte interaction has been discovered so far, many details of fertilization mechanism remain unknown. Both biochemical and biomechanical components exist in the fertilization process. Mammalian sperm evolved a ZP (zona pelucida) thrust reduction penetration strategy probably in response to the ZP resilient elasticity. Using a biomechanical approach and FEM analysis, local contact stress, ZP deformations during impact and attempt of sperm head penetration relative to different sperm impact angles (SIA) were studied. The sperm-oocyte contact was defined as non-linear frictional contact. A transient structural analysis at 37°C revealed that, from the mechanical standpoint there are SIA that are more favorable for possible ZP penetration due to larger equivalent stress of ZP. An "slip-stick" resembling effect was identified for almost all examined SIA. The sperm head-ZP contact area increases as SIA decreases. Favorable ZP-stress state for sperm penetration regarding SIA are discussed. PMID:26780648

  10. [Successful pregnancies after oocyte and embryo vitrification].

    PubMed

    Salazar, Francisco Hernández; Loza, Erik Omar Okhuysen; Lucas, Maria Teresa Huerta J; Gutiérrez, Gustavo Romero

    2008-02-01

    Cryopreservation of human oocytes represents a solution for ethic conflict about frozen embryo storage for patients with risk to develop ovarian hyperstimulation syndrome; also is an available technique to preserve fertility in women with cancer under treatment, in poor response patients, in case of premature ovarian failure or aging and for other medical or social conditions that require to delay pregnancies, as well as to make easier oocyte donation programs. This paper reports two cases of successful pregnancies after embryo and oocyte vitrification, as well as their results. The technique of vitrification with the cryotop method is an excellent alternative, efficient, fast and cheap for oocyte and embryo cryopreservation with high ranges of fertilization, cleavage and pregnancies with a normal evolution. PMID:18798404

  11. 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

  12. 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

  13. Metabolic Determinants of Mitochondrial Function in Oocytes.

    PubMed

    Seidler, Emily A; Moley, Kelle H

    2015-11-01

    Mitochondrial production of cellular energy is essential to oocyte function, zygote development and successful continuation of pregnancy. This review focuses on several key functions of healthy oocyte mitochondria and the effect of pathologic states such as aging, oxidative stress and apoptosis on these functions. The effect of these abnormal conditions is presented in terms of clinical presentations, specifically maternal obesity, diminished ovarian reserve and assisted reproductive technologies.

  14. Oocyte glutathione and fertilisation outcome of Macaca nemestrina and Macaca fascicularis in in vivo- and in vitro-matured oocytes

    PubMed Central

    Curnow, E. C.; Ryan, J. P.; Saunders, D. M.; Hayes, E. S.

    2010-01-01

    Fertilisation and development of IVM non-human primate oocytes is limited compared with that of in vivo-matured (IVO) oocytes. The present study describes the IVM of macaque oocytes with reference to oocyte glutathione (GSH). Timing of maturation, comparison of IVM media and cysteamine (CYS) supplementation as a modulator of GSH were investigated. A significantly greater proportion of oocytes reached MII after 30 h compared with 24 h of IVM. Following insemination, IVM oocytes had a significantly lower incidence of normal fertilisation (i.e. 2PN = two pronuclei and at least one polar body) and a higher rate of abnormal fertilisation (1PN = one pronucleus and at least one polar body) compared with IVO oocytes. Immunofluorescence of 1PN zygotes identified incomplete sperm head decondensation and failure of male pronucleus formation as the principal cause of abnormal fertilisation in IVM oocytes. The IVO oocytes had significantly higher GSH content than IVM oocytes. Cumulus-denuded oocytes had significantly lower GSH following IVM compared with immature oocytes at collection. Cysteamine supplementation of the IVM medium significantly increased the GSH level of cumulus-intact oocytes and reduced the incidence of 1PN formation, but did not improve GSH levels of the denuded oocyte. Suboptimal GSH levels in macaque IVM oocytes may be related to reduced fertilisation outcomes. PMID:20591337

  15. Oocyte glutathione and fertilisation outcome of Macaca nemestrina and Macaca fascicularis in in vivo- and in vitro-matured oocytes.

    PubMed

    Curnow, E C; Ryan, J P; Saunders, D M; Hayes, E S

    2010-01-01

    Fertilisation and development of IVM non-human primate oocytes is limited compared with that of in vivo-matured (IVO) oocytes. The present study describes the IVM of macaque oocytes with reference to oocyte glutathione (GSH). Timing of maturation, comparison of IVM media and cysteamine (CYS) supplementation as a modulator of GSH were investigated. A significantly greater proportion of oocytes reached MII after 30 h compared with 24 h of IVM. Following insemination, IVM oocytes had a significantly lower incidence of normal fertilisation (i.e. 2PN = two pronuclei and at least one polar body) and a higher rate of abnormal fertilisation (1PN = one pronucleus and at least one polar body) compared with IVO oocytes. Immunofluorescence of 1PN zygotes identified incomplete sperm head decondensation and failure of male pronucleus formation as the principal cause of abnormal fertilisation in IVM oocytes. The IVO oocytes had significantly higher GSH content than IVM oocytes. Cumulus-denuded oocytes had significantly lower GSH following IVM compared with immature oocytes at collection. Cysteamine supplementation of the IVM medium significantly increased the GSH level of cumulus-intact oocytes and reduced the incidence of 1PN formation, but did not improve GSH levels of the denuded oocyte. Suboptimal GSH levels in macaque IVM oocytes may be related to reduced fertilisation outcomes.

  16. Congenital Transmission of Experimental Leishmaniasis in a Hamster Model

    PubMed Central

    Osorio, Yaneth; Rodriguez, Luz D.; Bonilla, Diana L.; Peniche, Alex G.; Henao, Hector; Saldarriaga, Omar; Travi, Bruno L.

    2012-01-01

    Little information is available on transplacental transmission of Leishmania spp. We determined the frequency and impact of congenital infection caused by Leishmania panamensis or L. donovani in experimentally infected hamsters. A polymerase chain reaction showed that congenital transmission occurred in 25.8% (24 of 93) of offspring born to L. panamensis-infected hamsters and 14.6% (11 of 75) offspring born to L. donovani-infected hamsters. Mortality during lactation was higher in offspring born to L. panamensis-infected hamsters and offspring born to L. donovani-infected hamsters than controls, and lymphoproliferation to Leishmania was more frequent in offspring born to L. panamensis-infected hamsters (17.4%, 11 of 63) than in offspring born to L. donovani-infected hamsters (8.5%, 3 of 35). After weaning, only offspring born to L. donovani-infected hamsters had lower weight gain (P < 0.001) and hematocrit levels (P = 0.0045) than controls. Challenge of offspring born to L. panamensis-infected hamsters with L. panamensis showed no differences in lesion evolution, and offspring born to L. donovani-infected hamsters were more susceptible to L. donovani challenge than controls. Consequently, prenatal exposure of hamsters to L. donovani significantly increased the mortality risk and susceptibility to secondary homologous infection. PMID:22556079

  17. Asymmetric learning to avoid heterospecific males in Mesocricetus hamsters.

    PubMed

    delBarco-Trillo, Javier; Johnston, Robert E

    2012-08-01

    If a female mates with a male of a closely related species, her fitness is likely to decline. Consequently, females may develop behavioral mechanisms to avoid mating with heterospecific males. In some species, one such mechanism is for adult females to learn to discriminate against heterospecific males after exposure to such males. We have previously shown that adult, female Syrian hamsters (Mesocricetus auratus) learn to discriminate against male Turkish hamsters (Mesocricetus brandti) after exposure to a single heterospecific male during 8 days across a wire-mesh barrier. Here we repeated that experiment but this time we exposed female Turkish hamsters to a male Syrian hamster for 8 days and then measured sexual and aggressive behaviors towards that heterospecific male and towards a conspecific male. In contrast to female Syrian hamsters, female Turkish hamsters did not differ in their latency to go into lordosis or in any measure of aggression towards either type of male. Female Turkish hamsters spent less time in lordosis with the heterospecific male, but the percentage of trials in which females copulated with conspecific and heterospecific males did not differ. When comparing females from both species that had been exposed to a heterospecific male for 8days, female Syrian hamsters copulated less and were more aggressive towards the heterospecific male compared to the behavior of female Turkish hamsters. We discuss how this asymmetric response between females of the two species may be due to the much larger geographical range of Turkish hamsters compared to Syrian hamsters.

  18. Chemical penetration enhancers.

    PubMed

    Newton, Stephen J

    2013-01-01

    Chemical penetration enhancers are utilized in topical preparations as a method for enhancing permeation of drugs across the skin. In particular, they are utilized for transdermal delivery of medications in an attempt to produce a systemic response, to avoid first-pass metabolism, and to decrease the gastrointestinal transit time observed with oral medications. A review of the selection of chemical penetration enhancers, their mechanism of action, the most common chemical penetration enhancers in each class, and alternatives will be discussed in detail.

  19. Elastic and viscoelastic characterization of mouse oocytes using micropipette indentation.

    PubMed

    Liu, Xinyu; Shi, Jiayi; Zong, Zong; Wan, Kai-Tak; Sun, Yu

    2012-10-01

    This paper reports the first quantitative comparison study of elastic and viscoelastic properties of oocytes from young and aged mice. A force measurement technique, including a poly(dimethylsiloxane) (PDMS) cell holding device and a sub-pixel computer vision tracking algorithm, is utilized for measuring forces applied to an oocyte and resultant cell deformations in real time during oocyte manipulation. To characterize elastic and viscoelastic properties of the oocytes, a stress-relaxation indentation test is performed. A two-step, large-deformation mechanical model is developed to extract the mechanical properties of the oocytes from the measured force-deformation data. The experimental results demonstrate that the aged oocytes are significantly softer (instantaneous modulus: 2.2 vs. 5.2 kPa in young oocytes) but more viscous (relaxation time: 4.1 vs. 2.3 s in young oocytes) than the young oocytes.

  20. Elastic and viscoelastic characterization of mouse oocytes using micropipette indentation.

    PubMed

    Liu, Xinyu; Shi, Jiayi; Zong, Zong; Wan, Kai-Tak; Sun, Yu

    2012-10-01

    This paper reports the first quantitative comparison study of elastic and viscoelastic properties of oocytes from young and aged mice. A force measurement technique, including a poly(dimethylsiloxane) (PDMS) cell holding device and a sub-pixel computer vision tracking algorithm, is utilized for measuring forces applied to an oocyte and resultant cell deformations in real time during oocyte manipulation. To characterize elastic and viscoelastic properties of the oocytes, a stress-relaxation indentation test is performed. A two-step, large-deformation mechanical model is developed to extract the mechanical properties of the oocytes from the measured force-deformation data. The experimental results demonstrate that the aged oocytes are significantly softer (instantaneous modulus: 2.2 vs. 5.2 kPa in young oocytes) but more viscous (relaxation time: 4.1 vs. 2.3 s in young oocytes) than the young oocytes. PMID:22644532

  1. Preference for bedding material in Syrian hamsters.

    PubMed

    Lanteigne, M; Reebs, S G

    2006-10-01

    This study aimed to determine whether Syrian (golden) hamsters, Mesocricetus auratus, prefer certain bedding materials and whether bedding material can affect paw condition, body weight gain and wheel-running activity. In a first experiment, 26 male hamsters had access to two connected cages, each cage containing a different bedding material (either pine shavings, aspen shavings, corn cob or wood pellets). In a second experiment, 14 male hamsters had access to four connected cages that contained the different bedding materials and also a piece of paper towel to serve as nest material. In a third experiment, 30 male hamsters were each placed in a single cage, 10 of them with pine shavings, 10 with aspen shavings and 10 with corn cob, and they were monitored for 50 days. Significant preferences in the first experiment were: pine shavings over aspen shavings, corn cob over wood pellets, pine shavings over corn cob and aspen shavings over wood pellets (aspen shavings versus corn cob was not tested). However, there was no significant preference expressed in the second experiment, suggesting that the general preference for shavings in the first experiment was based on bedding material suitability as a nesting material. No significant effect of bedding material on paw condition, body weight gain and wheel-running activity was detected. None of the four bedding materials tested in this study can be judged to be inappropriate in the short term if nesting material is added to the cage and if the litter is changed regularly. PMID:17018212

  2. Follicular penetration and targeting.

    PubMed

    Lademann, Jürgen; Otberg, Nina; Jacobi, Ute; Hoffman, Robert M; Blume-Peytavi, Ulrike

    2005-12-01

    In the past, intercellular penetration was assumed to be the most important penetration pathway of topically applied substances. First hints that follicular penetration needs to be taken into consideration were confirmed by recent investigations, presented during the workshop "Follicular Penetration and Targeting" at the 4th Intercontinental Meeting of Hair Research Societies", in Berlin 2004. Hair follicles represent an efficient reservoir for the penetration of topically applied substances with subsequent targeting of distinct cell populations, e.g., nestin-expressing follicular bulge cells. The volume of this reservoir can be determined by differential stripping technology. The follicular penetration processes are significantly influenced by the state of the follicular infundibulum; recent experimental investigations could demonstrate that it is essential to distinguish between open and closed hair follicles. Topically applied substances can only penetrate into open hair follicle. Knowledge of follicular penetration is of high clinical relevance for functional targeting of distinct follicular regions. Human hair follicles show a hair-cycle-dependent variation of the dense neuronal and vascular network. Moreover, during hair follicle cycling with initiation of anagen, newly formed vessels occur. Thus, the potential of nestin-expressing hair follicle stem cells to form neurons and blood vessels was investigated.

  3. Session: Hard Rock Penetration

    SciTech Connect

    Tennyson, George P. Jr.; Dunn, James C.; Drumheller, Douglas S.; Glowka, David A.; Lysne, Peter

    1992-01-01

    This session at the Geothermal Energy Program Review X: Geothermal Energy and the Utility Market consisted of five presentations: ''Hard Rock Penetration - Summary'' by George P. Tennyson, Jr.; ''Overview - Hard Rock Penetration'' by James C. Dunn; ''An Overview of Acoustic Telemetry'' by Douglas S. Drumheller; ''Lost Circulation Technology Development Status'' by David A. Glowka; ''Downhole Memory-Logging Tools'' by Peter Lysne.

  4. Nuclear structures in Tribolium castaneum oocytes.

    PubMed

    Bogolyubov, Dmitry S; Batalova, Florina M; Kiselyov, Artyom M; Stepanova, Irina S

    2013-10-01

    The first ultrastructural and immunomorphological characteristics of the karyosphere (karyosome) and extrachromosomal nuclear bodies in the red flour beetle, Tribolium castaneum, are presented. The karyosphere forms early in the diplotene stage of meiotic prophase by the gathering of all oocyte chromosomes in a limited nuclear volume. Using the BrUTP assay, T. castaneum oocyte chromosomes united in the karyosphere maintain their transcriptional activity until the end of oocyte growth. Hyperphosphorylated RNA polymerase II and basal transcription factors (TFIID and TFIIH) were detected in the perichromatin region of the karyosphere. The T. castaneum karyosphere has an extrachromosomal capsule that separates chromosomes from the rest of the nucleoplasm. Certain structural proteins (F-actin, lamin B) were found in the capsule. Unexpectedly, the karyosphere capsule in T. castaneum oocytes was found to be enriched in TMG-capped snRNAs, which suggests that the capsule is not only a structural support for the karyosphere, but may be involved in biogenesis of snRNPs. We also identified the counterparts of 'universal' extrachromosomal nuclear domains, Cajal bodies (CBs) and interchromatin granule clusters (IGCs). Nuclear bodies containing IGC marker protein SC35 display some features unusual for typical IGCs. SC35 domains in T. castaneum oocytes are predominantly fibrillar complex bodies that do not contain trimethyl guanosine (TMG)-capped small nuclear (sn) RNAs. Microinjections of 2'-O-methyl (U)22 probes into the oocytes allowed revealing poly(A)+ RNAs in these nuclear domains. Several proteins related to mRNA export (heterogeneous ribonucleoprotein core protein A1, export adapters Y14 and Aly and export receptor NXF1) were also detected there. We believe that unusual SC35 nuclear domains of T. castaneum oocytes are possibly involved in mRNP but not snRNP biogenesis.

  5. Successful Cryopreservation of Mouse Oocytes by Using Low Concentrations of Trehalose and Dimethylsulfoxide1

    PubMed Central

    Eroglu, Ali; Bailey, Sarah E.; Toner, Mehmet; Toth, Thomas L.

    2008-01-01

    Sugars such as trehalose, sucrose, and glucose are effectively used by a variety of animals (e.g., brine shrimp, tardigrades, some frogs, and insects), as well as by bacteria, yeasts, and plant seeds to survive freezing and extreme drying. The objective of this study was to examine the potential application of sugars to mammalian oocyte cryopreservation. To this end, we used trehalose, a nonreducing disaccharide, and mouse metaphase II oocytes as models. Our experiments show that extracellular trehalose alone affords some protection at high subzero temperatures (e.g., −15°C), which diminishes with further cooling of the oocytes to −30°C and below. When present both intracellularly and extracellularly, trehalose dramatically improves the cryosurvival with increasing extracellular concentrations to 0.5 M, even after cooling to −196°C. Furthermore, the combination of intracellular and extracellular trehalose with small amounts of a conventional penetrating cryoprotectant (i.e., 0.5 M dimethylsulfoxide) provide high survival, fertilization, and embryonic development rates statistically similar to untreated controls. When transferred to foster mothers, cryopreserved oocytes give rise to healthy offspring showing the proof of principle. Our experiments with differential scanning calorimetry indicate that when cooled using the same cryopreservation protocol, the mixture of 0.5 M trehalose and cryopreservation medium undergoes glass transition at high subzero temperatures, which further substantiates the use of sugars as intracellular and extracellular cryoprotectants. Taken together, our results are in agreement with the survival schemes in nature and demonstrate the successful use of sugars in cryopreservation of mammalian oocytes. PMID:18815355

  6. 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

  7. Oocyte cryopreservation for donor egg banking.

    PubMed

    Cobo, Ana; Remohí, José; Chang, Ching-Chien; Nagy, Zsolt Peter

    2011-09-01

    Oocyte donation is an efficient alternative to using own oocytes in IVF treatment for different indications. Unfortunately, 'traditional' (fresh) egg donations are challenged with inefficiency, difficulties of synchronization, very long waiting periods and lack of quarantine measures. Given the recent improvements in the efficiency of oocyte cryopreservation, it is reasonable to examine if egg donation through oocyte cryopreservation has merits. The objective of the current manuscript is to review existing literature on this topic and to report on the most recent outcomes from two established donor cryobank centres. Reports on egg donation using slow freezing are scarce and though results are encouraging, outcomes are not yet comparable to a fresh egg donation treatment. Vitrification on the other hand appears to provide high survival rates (90%) of donor oocytes and comparable fertilization, embryo development, implantation and pregnancy rates to traditional (fresh) egg donation. Besides the excellent outcomes, the ease of use for both donors and recipients, higher efficiency, lower cost and avoiding the problem of synchronization are all features associated with the benefit of a donor egg cryobank and makes it likely that this approach becomes the future standard of care. Oocyte donation is one of the last resorts in IVF treatment for couples challenged with infertility problems. However, traditional (fresh) egg donation, as it is performed today, is not very efficient, as typically all eggs from one donor are given to only one recipient, it is arduous as it requires an excellent synchronization between the donor and recipient and there are months or years of waiting time. Because of the development of an efficient oocyte cryopreservation technique, it is now possible to cryo-store donor (as well as non-donor) eggs, maintaining their viability and allowing their use whenever there is demand. Therefore, creating a donor oocyte cryobank would carry many advantages

  8. Oocyte cryopreservation for donor egg banking.

    PubMed

    Cobo, Ana; Remohí, José; Chang, Ching-Chien; Nagy, Zsolt Peter

    2011-09-01

    Oocyte donation is an efficient alternative to using own oocytes in IVF treatment for different indications. Unfortunately, 'traditional' (fresh) egg donations are challenged with inefficiency, difficulties of synchronization, very long waiting periods and lack of quarantine measures. Given the recent improvements in the efficiency of oocyte cryopreservation, it is reasonable to examine if egg donation through oocyte cryopreservation has merits. The objective of the current manuscript is to review existing literature on this topic and to report on the most recent outcomes from two established donor cryobank centres. Reports on egg donation using slow freezing are scarce and though results are encouraging, outcomes are not yet comparable to a fresh egg donation treatment. Vitrification on the other hand appears to provide high survival rates (90%) of donor oocytes and comparable fertilization, embryo development, implantation and pregnancy rates to traditional (fresh) egg donation. Besides the excellent outcomes, the ease of use for both donors and recipients, higher efficiency, lower cost and avoiding the problem of synchronization are all features associated with the benefit of a donor egg cryobank and makes it likely that this approach becomes the future standard of care. Oocyte donation is one of the last resorts in IVF treatment for couples challenged with infertility problems. However, traditional (fresh) egg donation, as it is performed today, is not very efficient, as typically all eggs from one donor are given to only one recipient, it is arduous as it requires an excellent synchronization between the donor and recipient and there are months or years of waiting time. Because of the development of an efficient oocyte cryopreservation technique, it is now possible to cryo-store donor (as well as non-donor) eggs, maintaining their viability and allowing their use whenever there is demand. Therefore, creating a donor oocyte cryobank would carry many advantages

  9. Effect of N-acetyl-D-glucosamine on bovine sperm-oocyte interactions.

    PubMed

    Sakaguchi, Yosuke; Iwata, Hisataka; Kuwayama, Takehito; Monji, Yasunori

    2009-12-01

    N-Acetyl-D-glucosamine (GlcNAc) is a major component of glycosaminoglycan, which is involved in sperm-oocyte interactions. We examined the effect of adding GlcNAc and other monosaccharides, D-mannose and D-fucose, to the in vitro fertilization (IVF) medium on bovine sperm-oocyte interactions. In medium in which sperm and a zona pellucida (ZP) were co-incubated with monosaccharides for 5 min, addition of GlcNAc (5 or 25 mM) significantly reduced the number of sperm that attached to the ZP. Pretreatment of gametes with GlcNAc (5 mM) prior to co-incubation also suppressed sperm-ZP attachment. Addition of GlcNAc (5 or 25 mM) to the medium in which sperm and a ZP were co-incubated for 5 h, however, significantly increased the number of sperm binding to and penetrating the ZP in a concentration-related manner. The other monosaccharides, D-fucose and D-mannose, did not have this effect. Supplementation of the sperm-oocyte co-incubation medium with 5 mM GlcNAc also enhanced the rate of polyspermic fertilization. When the ZPs were removed from the oocytes, GlcNAc did not affect the fertilization rate. Furthermore, incubation of sperm with 5 mM GlcNAc induced sperm membrane destabilization and an acrosome reaction, as evidenced by the hypo-osmotic swelling test and fluorescein isothiocyanate-labeled peanut agglutinin/propidium iodide (FITC-PNA/PI) staining. Finally, GlcNAc suppressed ZP hardening following fertilization, as determined by measuring the time required for pronase to dissolve the ZP. In conclusion, supplementation of IVF medium with GlcNAc has various effects on sperm-oocyte interactions including suppression of initial attachment, induction of sperm membrane destabilization and acrosome reaction, increase in the number of sperm secondarily bound to and penetrating the ZP, suppression of ZP hardening following sperm-oocyte co-incubation and increase in the rate of polyspermic fertilization. PMID:19809222

  10. DNA damage response during mouse oocyte maturation.

    PubMed

    Mayer, Alexandra; Baran, Vladimir; Sakakibara, Yogo; Brzakova, Adela; Ferencova, Ivana; Motlik, Jan; Kitajima, Tomoya S; Schultz, Richard M; Solc, Petr

    2016-01-01

    Because low levels of DNA double strand breaks (DSBs) appear not to activate the ATM-mediated prophase I checkpoint in full-grown oocytes, there may exist mechanisms to protect chromosome integrity during meiotic maturation. Using live imaging we demonstrate that low levels of DSBs induced by the radiomimetic drug Neocarzinostatin (NCS) increase the incidence of chromosome fragments and lagging chromosomes but do not lead to APC/C activation and anaphase onset delay. The number of DSBs, represented by γH2AX foci, significantly decreases between prophase I and metaphase II in both control and NCS-treated oocytes. Transient treatment with NCS increases >2-fold the number of DSBs in prophase I oocytes, but less than 30% of these oocytes enter anaphase with segregation errors. MRE11, but not ATM, is essential to detect DSBs in prophase I and is involved in H2AX phosphorylation during metaphase I. Inhibiting MRE11 by mirin during meiotic maturation results in anaphase bridges and also increases the number of γH2AX foci in metaphase II.  Compromised DNA integrity in mirin-treated oocytes indicates a role for MRE11 in chromosome integrity during meiotic maturation. PMID:26745237

  11. 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.

  12. Bioactivation of diethylstilbestrol by the Syrian hamster kidney

    SciTech Connect

    Adams, S.P.

    1987-01-01

    Male Syrian golden hamsters chronically exposed to diethylstilbestrol (DES) develop renal adenocarcinomas with an incidence approaching 100%. The ability of the hamster kidney to bioactivate DES was assessed using hamster kidney slices. The male hamster renal cortex has a 2- to 5-fold greater capacity to irreversibly bind ({sup 3}H)DES as compared with female hamster renal cortex and with male hamster renal medulla. Incubation of the tissue under anaerobic conditions inhibited the metabolism and irreversible binding of ({sup 3}H)DES. Gel electrophoresis analysis of covalently modified proteins revealed several radioactive peaks indicating that specific adduct formation had occurred. The cytochrome P-450 inhibitors SKF 525-A, metyrapone, carbon monoxide, butylated hydroxytoluene, and dicumarol decreased the irreversible binding of ({sup 3}H)DES to renal cortical protein by 38 to 72%.

  13. Vitrification of buffalo (Bubalus bubalis) oocytes.

    PubMed

    Dhali, A; Manik, R S; Das, S K; Singla, S K; Palta, P

    2000-04-01

    The objective of the present study was to develop a method for the cryopreservation of buffalo oocytes by vitrification. Cumulus-oocyte complexes (COCs) were obtained from slaughterhouse ovaries. Prior to vitrification of COCs in the vitrification solution (VS) consisting of 4.5 M ethylene glycol, 3.4 M dimethyl sulfoxide, 5.56 mM glucose, 0.33 mM sodium pyruvate and 0.4% w/v bovine serum albumin in Dulbecco's phosphate buffered saline (DPBS), the COCs were exposed to the equilibration solution (50% VS v/v in DPBS) for 1 or 3 min at room temperature (25 to 30 degrees C). The COCs were then placed in 15-microL of VS and immediately loaded into 0.25-mL French straws, each containing 150 microL of 0.5 M sucrose in DPBS. The straws were placed in liquid nitrogen (LN2) vapor for 2 min, plunged and stored in LN2 for at least 7 d. The straws were thawed in warm water at 28 degrees C for 20 sec. For dilution, the COCs were equilibrated in 0.5 M sucrose in DPBS for 5 min and then washed 4 to 5 times in the washing medium (TCM-199+10% estrus buffalo serum). The proportion of oocytes recovered in a morphologically normal form was significantly higher (98 and 88%, respectively; P<0.05), and the proportion of oocytes recovered in a damaged form was significantly lower (2 and 12%, respectively; P<0.05) for the 3-min equilibration than for 1 min. For examining the in vitro developmental potential of vitrified-warmed oocytes, the oocytes were placed in 50-microL droplets (10 to 15 oocytes per droplet) of maturation medium (TCM-199+15% FBS+5 microg/mL FSH-P), covered with paraffin oil in a 35-mm Petri dish and cultured for 26 h in a CO2 incubator (5% CO2 in air) at 38.5 degrees C. Although the nuclear maturation rate did not differ between the 1- and 3-min equilibration periods (21.5+/-10.7 and 31.5+/-1.5%, respectively), the between-trial variation was very high for the 1-min period. This method of vitrification is simple and rapid, and can be useful for cryopreservation of

  14. Drinking, but not feeding, is opiate-sensitive in hamsters.

    PubMed

    Lowy, M T; Yim, G K

    1982-05-10

    The long-lasting opiate antagonist, naltrexone (NTX), was examined for its effects on various types of consummatory behavior in male golden hamsters and rats. Rat, but not hamster, 24 hr food and water intakes were significantly decreased by four daily NTX (10.0 mg/kg) injections. Hamsters displayed a minimal night to day feeding ratio compared to rats. Hamsters increased food intake following insulin (50 U/kg) administration, but not after 24 hr food deprivation (FD) or 2-deoxy-D-glucose (2-DG; 800 mg/kg) injections. NTX (1.0 and 10 mg/kg) had no effect on feeding, but markedly attenuated hamster drinking induced by 48 hr water deprivation or hypertonic saline injection. Dexamethasone (DEX), a glucocorticoid which depletes pituitary beta-endorphin and produces anorexia in rats, had no effect on daily hamster intake. Since the normal feeding profile of the hamster is similar to that of naloxone and DEX-treated rats, hamsters appear to lack an opiate-sensitive feeding system. In contrast, stimulated drinking behavior of hamsters operates through an opiate-sensitive mechanism. Thus, there are marked species differences concerning the involvement of endogenous opioids in consummatory behavior.

  15. SV40 induces mesotheliomas in hamsters.

    PubMed Central

    Cicala, C.; Pompetti, F.; Carbone, M.

    1993-01-01

    In the course of studies to elucidate the relative contribution of simian virus 40 (SV40) large T and small t proteins during oncogenesis, we observed the appearance of pericardial and pleural tumors in 100% of Syrian hamsters injected in the pleural space with wild type SV40. When SV40 was injected via the intracardiac or intraperitoneal routes, more than 50% of hamsters developed mesothelial tumors. Macroscopic, microscopic, ultramicroscopic, and histochemical characteristics identify these neoplasms and derived cell lines as mesotheliomas and mesothelioma-derived cell lines. The SV40 genome was integrated and expressed in the mesotheliomas and derived cell lines. The absence of mesotheliomas in hamsters injected with SV40 small t deletion mutants indicates that the small t protein plays an important role in the development of SV40-induced mesotheliomas. To the best of our knowledge, this is the first definitive report of virus-induced mesotheliomas in mammals. Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:8388174

  16. Molecular and immunological characterization of the first allergenic lipocalin in hamster: the major allergen from Siberian hamster (Phodopus sungorus).

    PubMed

    Torres, José Alberto; de Las Heras, Manuel; Maroto, Aroa Sanz; Vivanco, Fernando; Sastre, Joaquín; Pastor-Vargas, Carlos

    2014-08-22

    The most frequent pet allergy is to cat and dog, but in recent years, it has become increasingly popular to have other pets, and the risk of exposure to new allergens is more prevalent. The list of new pets includes hamsters, and one of the most popular hamsters is the Siberian hamster (Phodopus sungorus). The aim of this study was the characterization and cloning of the major allergen from this hamster. The study of its allergenicity and cross-reactivity could improve the specific diagnosis and treatment for hamster-allergic patients. Thirteen Siberian hamster-allergic patients were recruited at the outpatient clinic. Protein extracts were prepared from the hair, urine, and salivary glands of four hamster species (European, golden, Siberian, and Roborovski). IgE-binding proteins were detected by immunoblotting and identified by mass spectrometry. The recombinant protein was produced in Escherichia coli and then purified by metal chelate affinity chromatography. The allergenic properties of the recombinant protein were tested by ELISA and immunoblotting, and biological activity was tested according to capacity for basophil activation. Three IgE-binding proteins were identified in extracts obtained from Siberian hamster hair, urine, and salivary glands. All proteins corresponded to the same protein, which was identified as a lipocalin. This lipocalin had no cross-reactivity with common and golden hamsters. The recombinant allergen was cloned and purified, showing similar IgE reactivity in vitro to Siberian hamster protein extracts. Also, the recombinant allergen was capable of producing biological activation in vivo. The major Siberian hamster allergen was cloned, and allergenic properties were characterized, providing a new tool for specific diagnosis of allergy to Siberian hamster.

  17. Age-Associated Lipidome Changes in Metaphase II Mouse Oocytes

    PubMed Central

    Lee, Jae Won; Lee, Geun-Kyung; Suh, Chang Suk; Kim, Kwang Pyo; Lim, Hyunjung Jade

    2016-01-01

    The quality of mammalian oocytes declines with age, which negatively affects fertilization and developmental potential. The aging process often accompanies damages to macromolecules such as proteins, DNA, and lipids. To investigate if aged oocytes display an altered lipidome compared to young oocytes, we performed a global lipidomic analysis between oocytes from 4-week-old and 42 to 50-week-old mice. Increased oxidative stress is often considered as one of the main causes of cellular aging. Thus, we set up a group of 4-week-old oocytes treated with hydrogen peroxide (H2O2), a commonly used oxidative stressor, to compare if similar lipid species are altered between aged and oxidative-stressed oocytes. Between young and aged oocytes, we identified 26 decreased and 6 increased lipids in aged oocytes; and between young and H2O2-treated oocytes, we identified 35 decreased and 26 increased lipids in H2O2-treated oocytes. The decreased lipid species in these two comparisons were overlapped, whereas the increased lipid species were distinct. Multiple phospholipid classes, phosphatidic acid (PA), phosphatidylinositol (PI), phosphatidylserine (PS), and lysophosphatidylserine (LPS) significantly decreased both in H2O2-treated and aged oocytes, suggesting that the integrity of plasma membrane is similarly affected under these conditions. In contrast, a dramatic increase in diacylglycerol (DG) was only noted in H2O2-treated oocytes, indicating that the acute effect of H2O2-caused oxidative stress is distinct from aging-associated lipidome alteration. In H2O2-treated oocytes, the expression of lysophosphatidylcholine acyltransferase 1 increased along with increases in phosphatidylcholine. Overall, our data reveal that several classes of phospholipids are affected in aged oocytes, suggesting that the integrity of plasma membrane is associated with maintaining fertilization and developmental potential of mouse oocytes. PMID:26881843

  18. Artificial oocyte activation: evidence for clinical readiness.

    PubMed

    Ebner, T; Montag, M

    2016-03-01

    Artificial oocyte activation using Ca(2+)ionophores or similar compounds is a widely applied technique in IVF laboratories. This is all the more interesting as most of the agents aiming for intracellular Ca(2+) increase do not result in physiological Ca(2+) oscillations but much rather cause a single Ca(2+) transient. Two observations from mammals may explain why a rather non-physiological single Ca(2+) peak caused by ionophores is sufficient to rescue cycles showing severe male factor infertility, deficient oocyte maturation, developmental problems in humans, or both. On the one hand, it has been shown that it is mainly the initial Ca(2+) rise that drives further downstream events, in particular calcium/calmodulin-dependent protein kinase II (CaMKII) action, and on the other, it is possible that this enzyme remains active even in the absence of Ca(2+). It therefore seems that mammalian oocytes can respond to a wide range of intracellular Ca(2+) signals and have a surprisingly high degree of tolerance for changes in cytosolic Ca(2+). As epigenetic consequences or differences in gene expression have not been studied to date, artificial oocyte activation has to be considered as experimental and should only be applied with a proper indication. PMID:26776820

  19. [Karyosphere capsule in Tribolium castaneum oocytes].

    PubMed

    Batalova, F M; Bogoliubov, D S

    2013-01-01

    Structure and composition of the karyosphere (karyosome) capsule were studied in the oocytes of a laboratory insect, Tribolium castaneum, with the use of electron microscopy and immunoelectron cytochemistry. Basing on the study of nuclear structure dynamics, we distinguished 8 stages that characterize the period of oocyte growth. At the diplotene stage, T. castaneum oocyte chromosomes conjoin early into a compact karyosphere, but a significant chromatin condensation does not occur. The process of karyosphere formation is accompanied by the development of an extensive extrachromosome capsule surrounding chromatin. The capsule consists of a material of different morphological types. Significant molecular components of the T. castaneum karyosphere capsule are represented by the proteins of nuclear matrix including F-actin and lamin B. Besides the structural proteins, the Sm proteins of small nuclear (sn) RNPs and mature 2,2,7-trimethyl guanosine (TMG) 5'-capped snRNAs are revealed immunocytochemically in the karyosphere capsule. The obtained data can form a basis for further expansion of ideas on the functions of the karyosphere capsule as a specialized extrachromosomal nuclear domain of the oocytes. We believe that the T. castaneum karyosphere capsule plays not only a structural role, but may be involved directly in the processes related to gene expression.

  20. Calcium flux assay in Xenopus oocytes.

    PubMed

    Murphy, P M

    2001-05-01

    Many G protein-coupled receptors of interest to neuroscientists induce transient increases in [Ca(2+)](i), which can be used as a convenient measure of receptor activation in a variety of applications. This unit describes a simple calcium flux assay applied to Xenopus oocytes. PMID:18428482

  1. Calcium signaling differentiation during Xenopus oocyte maturation.

    PubMed

    El-Jouni, Wassim; Jang, Byungwoo; Haun, Shirley; Machaca, Khaled

    2005-12-15

    Ca(2+) is the universal signal for egg activation at fertilization in all sexually reproducing species. The Ca(2+) signal at fertilization is necessary for egg activation and exhibits specialized spatial and temporal dynamics. Eggs acquire the ability to produce the fertilization-specific Ca(2+) signal during oocyte maturation. However, the mechanisms regulating Ca(2+) signaling differentiation during oocyte maturation remain largely unknown. At fertilization, Xenopus eggs produce a cytoplasmic Ca(2+) (Ca(2+)(cyt)) rise that lasts for several minutes, and is required for egg activation. Here, we show that during oocyte maturation Ca(2+) transport effectors are tightly modulated. The plasma membrane Ca(2+) ATPase (PMCA) is completely internalized during maturation, and is therefore unable to extrude Ca(2+) out of the cell. Furthermore, IP(3)-dependent Ca(2+) release is required for the sustained Ca(2+)(cyt) rise in eggs, showing that Ca(2+) that is pumped into the ER leaks back out through IP(3) receptors. This apparent futile cycle allows eggs to maintain elevated cytoplasmic Ca(2+) despite the limited available Ca(2+) in intracellular stores. Therefore, Ca(2+) signaling differentiates in a highly orchestrated fashion during Xenopus oocyte maturation endowing the egg with the capacity to produce a sustained Ca(2+)(cyt) transient at fertilization, which defines the egg's competence to activate and initiate embryonic development.

  2. 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.

  3. Sperm-induced calcium oscillations of human oocytes show distinct features in oocyte center and periphery.

    PubMed

    Tesarik, J; Sousa, M; Mendoza, C

    1995-06-01

    Temporal and spatial characteristics of explosive periodic increases (spikes) of intracellular free Ca2+ concentration ([Ca2+]i) induced by sperm in human oocytes (Ca2+ oscillations) were analyzed by confocal laser scanning microscopy and compared to Ca2+ oscillations induced in oocytes by the thiol reagent thimerosal. During the steady-state period of sperm-induced Ca2+ oscillations, each individual [Ca2+]i spike invariably began from a focus in oocyte periphery and spread throughout the entire peripheral region before propagating to the central ooplasm. This peripheral Ca2+ wave was immediately followed by an explosive [Ca2+]i increase in the central ooplasm. However, this central [Ca2+]i rise only peaked when [Ca2+]i in the peripheral ooplasm was already on the decline. Moreover, the peak [Ca2+]i values were always considerably higher in the oocyte center than in the periphery. In contrast, thimerosal-induced Ca2+ oscillations did not show this particular form of propagation. These data show that sperm-induced Ca2+ oscillations have a unique pattern of spatial dynamics and suggest that the bulk of Ca2+ mobilized during each spike is released from stores that have a relatively high threshold for Ca(2+)-induced Ca2+ release (CICR). These stores are poorly developed, if not absent, in the oocyte cortex, and CICR from them is triggered by previous CICR from another type of store with a lower threshold that are preferentially located in the oocyte cortex and act as a detonator.

  4. Deployable Wireless Camera Penetrators

    NASA Technical Reports Server (NTRS)

    Badescu, Mircea; Jones, Jack; Sherrit, Stewart; Wu, Jiunn Jeng

    2008-01-01

    A lightweight, low-power camera dart has been designed and tested for context imaging of sampling sites and ground surveys from an aerobot or an orbiting spacecraft in a microgravity environment. The camera penetrators also can be used to image any line-of-sight surface, such as cliff walls, that is difficult to access. Tethered cameras to inspect the surfaces of planetary bodies use both power and signal transmission lines to operate. A tether adds the possibility of inadvertently anchoring the aerobot, and requires some form of station-keeping capability of the aerobot if extended examination time is required. The new camera penetrators are deployed without a tether, weigh less than 30 grams, and are disposable. They are designed to drop from any altitude with the boost in transmitting power currently demonstrated at approximately 100-m line-of-sight. The penetrators also can be deployed to monitor lander or rover operations from a distance, and can be used for surface surveys or for context information gathering from a touch-and-go sampling site. Thanks to wireless operation, the complexity of the sampling or survey mechanisms may be reduced. The penetrators may be battery powered for short-duration missions, or have solar panels for longer or intermittent duration missions. The imaging device is embedded in the penetrator, which is dropped or projected at the surface of a study site at 90 to the surface. Mirrors can be used in the design to image the ground or the horizon. Some of the camera features were tested using commercial "nanny" or "spy" camera components with the charge-coupled device (CCD) looking at a direction parallel to the ground. Figure 1 shows components of one camera that weighs less than 8 g and occupies a volume of 11 cm3. This camera could transmit a standard television signal, including sound, up to 100 m. Figure 2 shows the CAD models of a version of the penetrator. A low-volume array of such penetrator cameras could be deployed from an

  5. Penetration of yawed projectiles

    SciTech Connect

    Reaugh, J.E.

    1990-10-08

    We used computer simulations and experiment to study the penetration of tungsten-alloy projectiles into a thick, armored steel target. These projectiles, with length-to-diameter ratios of 4, strike the target with severe yaws, up to 90{degree}(side-on-impact), such as might be induced in an originally longer projectile by a multiple-spaced plate array. In this study, we focus on the terminal ballistics of these projectiles and ignore how the yaw was induced. We found that the minimum penetration depth occurs at 90{degree}yaw. This case is well approximated by the two-dimensional plane-strain penetration of a side-on cylinder. The ratio of penetration depth to diameter, P:D, for this case is larger than that for a sphere because the plane-strain geometry lacks hoop stress, which is activated in axisymmetric geometry. A more surprising result of work is that the penetration at 60{degree} yaw is only slightly deeper than that of the side-on impact. 8 refs., 15 figs., 3 tabs.

  6. In vitro maturation alters gene expression in bovine oocytes.

    PubMed

    Adona, Paulo R; Leal, Cláudia L V; Biase, Fernando H; De Bem, Tiago H; Mesquita, Lígia G; Meirelles, Flávio V; Ferraz, André L; Furlan, Luiz R; Monzani, Paulo S; Guemra, Samuel

    2016-08-01

    Gene expression profiling of in vivo- and in vitro-matured bovine oocytes can identify transcripts related to the developmental potential of oocytes. Nonetheless, the effects of in vitro culturing oocytes are yet to be fully understood. We tested the effects of in vitro maturation on the transcript profile of oocytes collected from Bos taurus indicus cows. We quantified the expression of 1488 genes in in vivo- and in vitro-matured oocytes. Of these, 51 genes were up-regulated, whereas 56 were down-regulated (≥2-fold) in in vivo-matured oocytes in comparison with in vitro-matured oocytes. Quantitative real-time polymerase chain reaction (PCR) of nine genes confirmed the microarray results of differential expression between in vivo- and in vitro-matured oocytes (EZR, EPN1, PSEN2, FST, IGFBP3, RBBP4, STAT3, FDPS and IRS1). We interrogated the results for enrichment of Gene Ontology categories and overlap with protein-protein interactions. The results revealed that the genes altered by in vitro maturation are mostly related to the regulation of oocyte metabolism. Additionally, analysis of protein-protein interactions uncovered two regulatory networks affected by the in vitro culture system. We propose that the differentially expressed genes are candidates for biomarkers of oocyte competence. In vitro oocyte maturation can affect the abundance of specific transcripts and are likely to deplete the developmental competence.

  7. Calcium signals and oocyte maturation in marine invertebrates.

    PubMed

    Deguchi, Ryusaku; Takeda, Noriyo; Stricker, Stephen A

    2015-01-01

    In various oocytes and eggs of animals, transient elevations in cytoplasmic calcium ion concentrations are known to regulate key processes during fertilization and the completion of meiosis. However, whether or not calcium transients also help to reinitiate meiotic progression at the onset of oocyte maturation remains controversial. This article summarizes reports of calcium signals playing essential roles during maturation onset (=germinal vesicle breakdown, GVBD) in several kinds of marine invertebrate oocytes. Conversely, other data from the literature, as well as previously unpublished findings for jellyfish oocytes, fail to support the view that calcium signals are required for GVBD. In addition to assessing the effects of calcium transients on GVBD in marine invertebrate oocytes, the ability of maturing oocytes to enhance their calcium-releasing capabilities after GVBD is also reviewed. Furthermore, possible explanations are proposed for the contradictory results that have been obtained regarding calcium signals during oocyte maturation in marine invertebrates. PMID:26679945

  8. Mitofusin-2 is required for mouse oocyte meiotic maturation

    PubMed Central

    Zhang, Jing-Hua; Zhang, Teng; Gao, Si-Hua; Wang, Ke; Yang, Xiu-Yan; Mo, Fang-Fang; Na Yu; An, Tian; Li, Yu-Feng; Hu, Ji-Wei; Jiang, Guang-Jian

    2016-01-01

    Mitofusin-2 (Mfn2) is essential for embryonic development, anti-apoptotic events, protection against free radical-induced lesions, and mitochondrial fusion in many cells. However, little is known about its mechanism and function during oocyte maturation. In this study, we found that Mfn2 was expressed in the cytoplasm during different stages of mouse oocyte maturation. Mfn2 was mainly associated with α-tubulin during oocyte maturation. Knockdown of Mfn2 by specific siRNA injection into oocytes caused the mitochondrial morphology and quantity to change, resulting in severely defective spindles and misaligned chromosomes. This led to metaphase I arrest and the failure of first polar body extrusion. Furthermore, Mfn2 depletion from GV stage oocytes caused the redistribution of p38 MAPK in oocyte cytoplasm. These findings provide insights into potential mechanisms of Mfn2-mediated cellular alterations, which may have significant implications for oocyte maturation. PMID:27485634

  9. SIRT1, 2, 3 protect mouse oocytes from postovulatory aging.

    PubMed

    Zhang, Teng; Zhou, Yang; Li, Li; Wang, Hong-Hui; Ma, Xue-Shan; Qian, Wei-Ping; Shen, Wei; Schatten, Heide; Sun, Qing-Yuan

    2016-04-01

    The quality of metaphase II oocytes will undergo a time-dependent deterioration following ovulation as the result of the oocyte aging process. In this study, we determined that the expression of sirtuin family members (SIRT1, 2, 3) was dramatically reduced in mouse oocytes aged in vivo or in vitro. Increased intracellular ROS was observed when SIRT1, 2, 3 activity was inhibited. Increased frequency of spindle defects and disturbed distribution of mitochondria were also observed in MII oocytes aged in vitro after treatment with Nicotinamide (NAM), indicating that inhibition of SIRT1, 2, 3 may accelerate postovulatory oocyte aging. Interestingly, when MII oocytes were exposed to caffeine, the decline of SIRT1, 2, 3 mRNA levels was delayed and the aging-associated defective phenotypes could be improved. The results suggest that the SIRT1, 2, 3 pathway may play a potential protective role against postovulatory oocyte aging by controlling ROS generation. PMID:26974211

  10. Calcium signals and oocyte maturation in marine invertebrates.

    PubMed

    Deguchi, Ryusaku; Takeda, Noriyo; Stricker, Stephen A

    2015-01-01

    In various oocytes and eggs of animals, transient elevations in cytoplasmic calcium ion concentrations are known to regulate key processes during fertilization and the completion of meiosis. However, whether or not calcium transients also help to reinitiate meiotic progression at the onset of oocyte maturation remains controversial. This article summarizes reports of calcium signals playing essential roles during maturation onset (=germinal vesicle breakdown, GVBD) in several kinds of marine invertebrate oocytes. Conversely, other data from the literature, as well as previously unpublished findings for jellyfish oocytes, fail to support the view that calcium signals are required for GVBD. In addition to assessing the effects of calcium transients on GVBD in marine invertebrate oocytes, the ability of maturing oocytes to enhance their calcium-releasing capabilities after GVBD is also reviewed. Furthermore, possible explanations are proposed for the contradictory results that have been obtained regarding calcium signals during oocyte maturation in marine invertebrates.

  11. SIRT1, 2, 3 protect mouse oocytes from postovulatory aging

    PubMed Central

    Zhang, Teng; Zhou, Yang; Li, Li; Wang, Hong-Hui; Ma, Xue-Shan; Qian, Wei-Ping; Shen, Wei; Schatten, Heide; Sun, Qing-Yuan

    2016-01-01

    The quality of metaphase II oocytes will undergo a time-dependent deterioration following ovulation as the result of the oocyte aging process. In this study, we determined that the expression of sirtuin family members (SIRT1, 2, 3) was dramatically reduced in mouse oocytes aged in vivo or in vitro. Increased intracellular ROS was observed when SIRT1, 2, 3 activity was inhibited. Increased frequency of spindle defects and disturbed distribution of mitochondria were also observed in MII oocytes aged in vitro after treatment with Nicotinamide (NAM), indicating that inhibition of SIRT1, 2, 3 may accelerate postovulatory oocyte aging. Interestingly, when MII oocytes were exposed to caffeine, the decline of SIRT1, 2, 3 mRNA levels was delayed and the aging-associated defective phenotypes could be improved. The results suggest that the SIRT1, 2, 3 pathway may play a potential protective role against postovulatory oocyte aging by controlling ROS generation. PMID:26974211

  12. Proteasomal degradation of ubiquitinated proteins in oocyte meiosis and fertilization in mammals.

    PubMed

    Karabinova, Pavla; Kubelka, Michal; Susor, Andrej

    2011-10-01

    Gametogenesis and fertilization are the key events in sexual reproduction. In the female, meiosis results in a large oocyte that is competent for fertilization and fundamental for the success of early embryonic development. Progression through meiosis is monitored by fine regulatory mechanisms. In this review, we focus on one of the most well-known regulatory elements, the E3 ligase APC/C, which mediates proteolytic degradation of a number of important substrates via the ubiquitin proteasome pathway (UPP). The UPP also indirectly regulates protein synthesis by affecting proteins involved in RNA metabolism, a process that is paramount for the transcriptionally silent oocyte. During the past few years, more evidence has accumulated to suggest that the UPP has an important role in zona pellucida penetration and gamete fusion in mammals. This review focuses on the function of the UPP in regulating oocyte meiotic maturation in mammals, with special attention to its role in chromosome segregation and polar body extrusion, its role in the acquisition of meiotic/developmental competence and recent advances in our understanding of the UPP role in fertilization.

  13. Signal transduction in mammalian oocytes during fertilization.

    PubMed

    Machaty, Zoltan

    2016-01-01

    Mammalian embryo development begins when the fertilizing sperm triggers a series of elevations in the oocyte's intracellular free Ca(2+) concentration. The elevations are the result of repeated release and re-uptake of Ca(2+) stored in the smooth endoplasmic reticulum. Ca(2+) release is primarily mediated by the phosphoinositide signaling system of the oocyte. The system is stimulated when the sperm causes the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) into inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG); IP3 then binds its receptor on the surface of the endoplasmic reticulum that induces Ca(2+) release. The manner in which the sperm generates IP3, the Ca(2+) mobilizing second messenger, has been the subject of extensive research for a long time. The sperm factor hypothesis has eventually gained general acceptance, according to which it is a molecule from the sperm that diffuses into the ooplasm and stimulates the phosphoinositide cascade. Much evidence now indicates that the sperm-derived factor is phospholipase C-zeta (PLCζ) that cleaves PIP2 and generates IP3, eventually leading to oocyte activation. A recent addition to the candidate sperm factor list is the post-acrosomal sheath WW domain-binding protein (PAWP), whose role at fertilization is currently under debate. Ca(2+) influx across the plasma membrane is also important as, in the absence of extracellular Ca(2+), the oscillations run down prematurely. In pig oocytes, the influx that sustains the oscillations seems to be regulated by the filling status of the stores, whereas in the mouse other mechanisms might be involved. This work summarizes the current understanding of Ca(2+) signaling in mammalian oocytes.

  14. Transformation of Hamster Embryo Cells and Tumor Induction in Newborn Hamsters by Simian Adenovirus SV11

    PubMed Central

    Casto, Bruce C.

    1969-01-01

    Simian adenovirus, SV11, readily transformed hamster embryo cell cultures in vitro and produced tumors in vivo when inoculated into newborn hamsters. Foci consisting of small, loosely attached, rounded cells could be seen as early as 7 days postinoculation. Many of these cells contained several nuclei or the nucleus was multilobed. The cells grew without extensive cell to cell contact or formed small chains or clusters when passaged in vitro. This pattern of cell morphology and growth has not been reported with other simian or human adenovirus-transformed cells. Linearity of foci formation with virus dilution was observed when the virus multiplicity was less than 3 plaque-forming units (PFU)/cell. The PFU to focus-forming units ratio for SV11 was found to be 2 × 104 to 4 × 104, which is approximately 5- to 10-fold and 50- to 100-fold lower than those reported for simian adenovirus, SA7, and human adenovirus type 12, respectively. Cells transformed by SV11: (i) produced tumors when inoculated into young hamsters, (ii) contained tumor antigen which reacts with serum obtained from hamsters bearing SV11 passaged tumors, and (iii) could be propagated in vitro through an indefinite number of generations. Images PMID:5786181

  15. Characteristics of 263K Scrapie Agent in Multiple Hamster Species

    PubMed Central

    Barbian, Kent D.; Race, Brent; Favara, Cynthia; Gardner, Don; Taubner, Lara; Porcella, Stephen; Race, Richard

    2009-01-01

    Transmissible spongiform encephalopathy (TSE) diseases are known to cross species barriers, but the pathologic and biochemical changes that occur during transmission are not well understood. To better understand these changes, we infected 6 hamster species with 263K hamster scrapie strain and, after each of 3 successive passages in the new species, analyzed abnormal proteinase K (PK)–resistant prion protein (PrPres) glycoform ratios, PrPres PK sensitivity, incubation periods, and lesion profiles. Unique 263K molecular and biochemical profiles evolved in each of the infected hamster species. Characteristics of 263K in the new hamster species seemed to correlate best with host factors rather than agent strain. Furthermore, 2 polymorphic regions of the prion protein amino acid sequence correlated with profile differences in these TSE-infected hamster species. PMID:19193264

  16. Oocyte induction of EGF responsiveness in somatic cells is associated with the acquisition of porcine oocyte developmental competence.

    PubMed

    Ritter, Lesley J; Sugimura, Satoshi; Gilchrist, Robert B

    2015-06-01

    Oocytes progressively acquire the competence to support embryo development as oogenesis proceeds with ovarian folliculogenesis. The objectives of this study were to investigate oocyte-secreted factor (OSF) participation in the development of somatic cell epidermal growth factor (EGF) responsiveness associated with oocyte developmental competence. A well-established porcine model was employed using oocytes from small (<4 mm) vs medium sized (>4 mm) antral follicles, representing low vs moderate developmental competence, respectively. Cumulus-oocyte complexes (COCs) were treated in vitro with inducers of oocyte maturation, and cumulus cell functions and oocyte developmental competence were assessed. COCs from small follicles responded to FSH but, unlike COCs from larger follicles, were incapable of responding to EGF family growth factors known to mediate oocyte maturation in vivo, exhibiting perturbed cumulus expansion and expression of associated transcripts (HAS2 and TNFAIP6). Low and moderate competence COCs expressed equivalent levels of EGF receptor (EGFR) mRNA; however, the former had less total EGFR protein leading to failed activation of phospho-EGFR and phospho-ERK1/2, despite equivalent total ERK1/2 protein levels. Native OSFs from moderate, but not from low, competence oocytes established EGF responsiveness in low competence COCs. Four candidate recombinant OSFs failed to mimic the actions of native OSFs in regulating cumulus expansion. Treatment with OSFs and EGF enhanced oocyte competence but only of the low competence COCs. These data suggest that developmental acquisition by the oocyte of capacity to regulate EGF responsiveness in the oocyte's somatic cells is a major milestone in the oocyte's developmental program and contributes to coordinated oocyte and somatic cell development.

  17. Circadian rhythms of photorefractory siberian hamsters remain responsive to melatonin.

    PubMed

    Butler, Matthew P; Paul, Matthew J; Turner, Kevin W; Park, Jin Ho; Driscoll, Joseph R; Kriegsfeld, Lance J; Zucker, Irving

    2008-04-01

    Short day lengths increase the duration of nocturnal melatonin (Mel) secretion, which induces the winter phenotype in Siberian hamsters. After several months of continued exposure to short days, hamsters spontaneously revert to the spring-summer phenotype. This transition has been attributed to the development of refractoriness of Mel-binding tissues, including the suprachiasmatic nucleus (SCN), to long-duration Mel signals. The SCN of Siberian hamsters is required for the seasonal response to winter-like Mel signals, and becomes refractory to previously effective long-duration Mel signals restricted to this area. Acute Mel treatment phase shifts circadian locomotor rhythms of photosensitive Siberian hamsters, presumably by affecting circadian oscillators in the SCN. We tested whether seasonal refractoriness of the SCN to long-duration Mel signals also renders the circadian system of Siberian hamsters unresponsive to Mel. Males manifesting free-running circadian rhythms in constant dim red light were injected with Mel or vehicle for 5 days on a 23.5-h T-cycle beginning at circadian time 10. Mel injections caused significantly larger phase advances in activity onset than did the saline vehicle, but the magnitude of phase shifts to Mel did not differ between photorefractory and photosensitive hamsters. Similarly, when entrained to a 16-h light/8-h dark photocycle, photorefractory and photosensitive hamsters did not differ in their response to Mel injected 4 h before the onset of the dark phase. Activity onset in Mel-injected hamsters was masked by light but was revealed to be significantly earlier than in vehicle-injected hamsters upon transfer to constant dim red light. The acute effects of melatonin on circadian behavioral rhythms are preserved in photorefractory hamsters.

  18. Complementary histological and genomic analyses reveal marked differences in the developmental trajectories of ovaries in Siberian hamsters raised in long- and short-day lengths.

    PubMed

    Park, Sung-Un; Bernstein, Adrien N; Place, Ned J

    2014-03-01

    Siberian hamsters (Phodopus sungorus) delay sexual development when raised in short-day (SD; 10 hr light: 14 hr dark) conditions, which leads to delayed onset of estrous cycles and ovulations as compared to females raised in long-day (LD; 16 hr light: 8 hr dark) conditions. In addition to the absence of pre-ovulatory follicles and corpora lutea, the ovaries of SD-reared Siberian hamsters are characterized by an abundance of hypertrophied granulosa cells (HGCs) that surround atretic oocytes. To determine the age at which the histology of LD and SD ovaries first diverge, including the initial appearance of HGCs in SD conditions, we examined hamster ovaries histologically at 1, 2, 3, 4, 6, 8, 10, and 12 weeks of age. After identifying subtle differences in LD and SD ovarian histology at 4 weeks of age, we searched for differences in ovarian gene expression at 3 and 8 weeks of age, which correspond to the ages when ovarian histology do not differ (3 weeks) versus the earliest age when HGCs were observed (8 weeks). At 3 weeks, only 14 genes were differentially expressed in LD and SD ovaries, whereas 183 genes were differentially expressed at 8 weeks. Overall, our findings demonstrate that ovarian development under SD conditions is not simply arrested at an early stage of LD development, but rather utilizes a developmental path that is distinct from that used in LD ovaries.

  19. Chainsaw penetrating neck injury.

    PubMed

    Brown, A F

    1995-06-01

    A case of chainsaw injury to the neck is described. Previous reports in the English language are exceedingly rare. A brief discussion of safety features on chain saws is followed by a review of selective vs. mandatory surgical exploration in penetrating neck trauma, including the role of ancillary diagnostic tests.

  20. Penetration resistant barrier

    DOEpatents

    Hoover, William R.; Mead, Keith E.; Street, Henry K.

    1977-01-01

    The disclosure relates to a barrier for resisting penetration by such as hand tools and oxy-acetylene cutting torches. The barrier comprises a layer of firebrick, which is preferably epoxy impregnated sandwiched between inner and outer layers of steel. Between the firebrick and steel are layers of resilient rubber-like filler.

  1. Jet penetration in glass

    SciTech Connect

    Moran, B.; Glenn, L.A.; Kusubov, A.

    1991-05-01

    We describe a phenomenological model which accounts for the mechanical response of glass to intense impulsive loading. An important aspect of this response is the dilatancy accompanying fracture. We have also conducted a number of experiments with 38.1-mm diameter precision shaped charges to establish the performance against various targets and to allow evaluation of our model. At 3 charge diameters standoff, the data indicate that both virgin and damaged glass offer better (Bernoulli-scaled) resistance to penetration than either of 4340 steel, or 6061-T6 aluminum alloy. Time-resolved measurements indicate two distinct phases of jet penetration in glass: An initial hydrodynamic phase, and a second phase characterized by a slower penetration velocity. Our calculations show that at early time, a crater is formed around the jet and only the tip of the undisturbed jet interacts with the glass. At late time the glass has collapsed on the jet and degraded penetration continues via a disturbed and fragmented jet.

  2. Tumor-Penetrating Peptides

    PubMed Central

    Teesalu, Tambet; Sugahara, Kazuki N.; Ruoslahti, Erkki

    2013-01-01

    Tumor-homing peptides can be used to deliver drugs into tumors. Phage library screening in live mice has recently identified homing peptides that specifically recognize the endothelium of tumor vessels, extravasate, and penetrate deep into the extravascular tumor tissue. The prototypic peptide of this class, iRGD (CRGDKGPDC), contains the integrin-binding RGD motif. RGD mediates tumor-homing through binding to αv integrins, which are selectively expressed on various cells in tumors, including tumor endothelial cells. The tumor-penetrating properties of iRGD are mediated by a second sequence motif, R/KXXR/K. This C-end Rule (or CendR) motif is active only when the second basic residue is exposed at the C-terminus of the peptide. Proteolytic processing of iRGD in tumors activates the cryptic CendR motif, which then binds to neuropilin-1 activating an endocytic bulk transport pathway through tumor tissue. Phage screening has also yielded tumor-penetrating peptides that function like iRGD in activating the CendR pathway, but bind to a different primary receptor. Moreover, novel tumor-homing peptides can be constructed from tumor-homing motifs, CendR elements and protease cleavage sites. Pathologies other than tumors can be targeted with tissue-penetrating peptides, and the primary receptor can also be a vascular “zip code” of a normal tissue. The CendR technology provides a solution to a major problem in tumor therapy, poor penetration of drugs into tumors. The tumor-penetrating peptides are capable of taking a payload deep into tumor tissue in mice, and they also penetrate into human tumors ex vivo. Targeting with these peptides specifically increases the accumulation in tumors of a variety of drugs and contrast agents, such as doxorubicin, antibodies, and nanoparticle-based compounds. Remarkably the drug to be targeted does not have to be coupled to the peptide; the bulk transport system activated by the peptide sweeps along any compound that is present in the

  3. Sex aneuploidy of unfertilized human oocytes after intracytoplasmic sperm injection

    SciTech Connect

    Lee, G.; Ward, D.C.; Jones, E.E.

    1994-09-01

    Intracytoplasmic sperm injection (ICSI) has recently achieved successful fertilization and pregnancy in human in vitro fertilization, particularly in cases of severe male factor infertility. One criticism of this novel clinical technique is that it bypasses the natural selection process of fertilization. We use fluorescence in situ hybridization (FISH) to analyze oocytes which fail to fertilize after ICSI in the Yale IVF Program. The purpose of this study is to determine whether failed fertilization after ICSI can be attributed to sex chromosome aneuploidy in the oocyte. Fertilization of oocytes is determined by the presence of two pronuclei on light microscopic examination (400X). Multi-probe FISH with DAPI (4,6,-diamino-2-phenyl-indole) counterstain is then performed to determine oocyte ploidy and the presence of decondensed sperm. Centromeric probes for X, Y and 17 are used simultaneously in each oocyte for in situ hybridization to oocyte chromatin. In all oocytes examined after ICSI to date, unfertilized oocytes have decondensed sperm DNA present confirming appropriate intracytoplasmic placement of the sperm. Preliminary results obtained from 31 oocytes have not identified any sex chromosome aneuploidies. The FISH technique used in post-ICSI oocytes is a model system for delineating genetic causes of failed fertilization in the human.

  4. Ultrastructural observation of oocytes in six types of stony corals.

    PubMed

    Tsai, Sujune; Chang, Wei-Chieh; Chavanich, Suchana; Viyakarn, Voranop; Lin, Chiahsin

    2016-08-01

    In this study, the ultrastructure of the oocytes of 6 types of scleractinian corals was observed by transmission electron microscopy (TEM). Moreover, histological and ultrastructural analyses were performed to improve our understanding of the organelles involved in coral oocyte formation. In all 6 stony coral species, the microvilli were tubular and directly grew from the surface of the oocyte membrane; yolk bodies, lipid granules, and cortical alveoli accounted for most of the volume inside the oocytes, suggesting that they are associated with energy storage and buoyancy. Clear differences were observed in the size of yolk bodies and lipid granules in the oocytes of the 6 stony coral species, which occupied approximately 55%-80% of the inner space of the oocytes. Galaxea fascicularis exhibited the largest lipid granule volume, but the oocytes contained only an average number of 12.45 lipid granules per unit area. Only Montipora incrassata oocytes contained symbiotic algae. The smallest size and proportion of lipid granules in M. incrassata oocytes may be attributed to the presence of symbiotic algae and large yolk bodies, which may help oocytes produce energy and function as a nutritional source. This study is crucial for improving the understanding of the basic biology of coral reproduction, and the ensuing datasets is critical for conservation-oriented studies seeking to cryopreserve corals during these times of dramatic global climate change. PMID:27265208

  5. Accurate dispensing system for single oocytes using air ejection

    PubMed Central

    Feng, Lin; Sun, Yiling; Ohsumi, Chisato; Arai, Fumihito

    2013-01-01

    In this study, we propose a new approach to increase the success rate of single-oocyte dispensing and investigate the subsequent viability of the dispensed oocytes. We used a pair of capacitance sensors placed in a microfluidic chip to detect the oocyte, and custom-designed a special buffer zone in the microchannel to decelerate the flow velocity and reduce the hydraulic pressure acting on the oocyte. In the buffer zone, a semicircular bay, formed by equally spaced micro-pillars, is used to stop the oocyte at the dispensing nozzle hole. Finally, the oocyte is ejected by airflow to the culture array. The novel feature of the developed microfluidic system is that the extraordinary improvement in success rate is accompanied by a lack of change in oocyte survival rate (as assessed by a comparison of survival rates before and after the dispensing procedure). By using this device, we achieved a highly accurate single-oocyte dispensing process with a success rate of 100%. The oocyte survival rate is approximately 70%, regardless of whether or not the oocyte is dispensed. The newly proposed system has the advantages of high operation speed and potential usage for two-dimensional micropatterning. PMID:24404076

  6. Follicle environment and quality of in vitro matured oocytes.

    PubMed

    Sirard, Marc-André

    2011-06-01

    In mammalian reproduction, the oocyte depends on the ovarian follicle for most of its growth. They form a bipolar partnership and the status of one will impact the functioning of the other. When oocytes are removed from their follicle by ovulation, they have normally completed all the steps required to begin their journey into the oviduct and drive the early embryonic development. When oocytes are removed from their follicle before natural ovulation, the process by which they acquire all the important components for their journey might not be completed and their ability to mature, fertilize or develop into embryos or to term might be compromised. Animal models have been useful to define the important steps required for the oocyte's growth phase, and in the mouse, when the oocyte has reached its full size, the program is ready. This is not the case in larger mammals where the completion of growth does not ensure that the oocyte is fully capable of undergoing all the steps to the embryo and to term. The final steps of oocyte preparation also involve a progressive condensation of the chromatin that may facilitate normal maturation but may also indirectly reduce the lifespan of the oocyte. In such a scenario, the oocyte would have an expiration date when fully competent. In humans, a number of indications may justify the aspiration of oocytes from unstimulated patients and the development of an in vitro maturation (IVM) process that would allow fertilization and subsequent development. This objective could be realized by a better understanding of the essential follicular contribution required before removing the oocyte. Therefore, this review will focus on the large animal models where IVM has been used and studied for more than 25 years. The status of the follicle at the time of oocyte recovery and the status of the oocyte's chromatin will be described in detail as they have a significant impact on the outcome.

  7. Role of arachidonic acid cascade in Rhinella arenarum oocyte maturation.

    PubMed

    Ortiz, Maria Eugenia; Arias-Torres, Ana Josefina; Zelarayán, Liliana Isabel

    2015-08-01

    There are no studies that document the production of prostaglandins (PGs) or their role in Rhinella arenarum oocyte maturation. In this study, we analysed the effect of arachidonic acid (AA) and prostaglandins (PGs) on maturation, activation and pronuclear formation in R. arenarum oocytes. Our results demonstrated that AA was capable of inducing maturation in time-dependent and dose-dependent manner. Arachidonic acid-induced maturation was inhibited by indomethacin. PGs from AA hydrolysis, such as prostaglandin F2α (PGF2α) and, to a lesser extent, PGE2, induced meiosis resumption. Oocyte maturation in response to PGF2α was similar to that produced by progesterone (P4). Oocyte response to PGE1 was scarce. Rhinella arenarum oocyte PGF2α-induced maturation showed seasonal variation. From February to June, oocytes presented low sensitivity to PGF2α. In following periods, this response increased until a maximum was reached during October to January, a close temporal correlation with oocyte response to P4 being observed. The effect of PGF2α on maturation was verified by analysing the capacity of oocytes to activate and form pronuclei after being injected with homologous sperm. The cytological analysis of activated oocytes demonstrated the absence of cortical granules in oocytes, suggesting that PGF2α induces germinal vesicle breakdown (GVBD) and meiosis resumption up to metaphase II. In turn, oocytes matured by the action of PGF2α were able to form pronuclei after fertilization in a similar way to oocyte maturated by P4. In microinjection of mature cytoplasm experiments, the transformation of pre-maturation promoting factor (pre-MPF) to MPF was observed when oocytes were treated with PGF2α. In summary, our results illustrated the participation of the AA cascade and its metabolites in maturation, activation and pronuclei formation in R. arenarum. PMID:24964276

  8. A new rolling culture-based in vitro fertilization system capable of reducing polyspermy in porcine oocytes.

    PubMed

    Kitaji, Hideki; Ookutsu, Shoji; Sato, Masahiro; Miyoshi, Kazuchika

    2015-05-01

    The high incidence of polyspermy is one of the major obstacles during in vitro fertilization (IVF) in pigs. To overcome this, we developed a novel IVF method, which involves constant rotation. Oocytes matured in vitro were mixed with spermatozoa (0.2 × 10(5) sperm/mL) in an IVF medium (200 μL) using a 200 μL PCR tube. This tube was then rotated at 1 rpm for 6 h at 38.5°C in a rotation mixer (experimental group). A second PCR tube was simultaneously cultured without rotation (control group). The rate of polyspermy was evaluated 12 h after insemination and was significantly (P < 0.05; 21.0% vs. 48.3%) lower in the experimental group than in the control group. Sperm penetration rate was similar in oocytes from the experimental and control groups (75.2% vs. 83.1%). However, monospermic fertilization rate of the oocytes was significantly (P < 0.05; 44.8% vs. 21.2%) higher in the experimental group than in the control group. Furthermore, the rate of blastocyst formation (30.1% vs. 20.8%) increased in the experimental group, as compared to the control group. This present system will contribute to increase the efficacy of blastocyst production through reduction of polyspermic penetration.

  9. Vitrification of oocytes, embryos and blastocysts.

    PubMed

    Mukaida, Tetsunori; Oka, Chikahiro

    2012-12-01

    In assisted reproductive technology, cryopreservation of human oocytes and embryos has been significantly improved by refined slow-cooling and the new vitrification method. The slow-cooling method requires a programmed cryo-machine, and usually takes several hours. It is, however, difficult to eliminate injuries resulting from ice formation completely. Vitrification has become a reliable strategy because it is simple, can lead to high survival rates and viability, and has better clinical outcome. Vitrification transforms cells into an amorphous glassy state inside and outside the vitrified cell with ultra-rapid cooling and warming steps by plunging the oocytes and embryos into liquid nitrogen, instead of ice-crystal formation. Over the past decade, several advances in vitrification technologies have improved clinical efficiency and outcome. In this chapter, we focus on vitrification technologies for cryopreservation in human assisted reproductive technology. PMID:22940094

  10. An antisperm monoclonal antibody inhibits sperm fusion with zona-free hamster eggs but not homologous eggs.

    PubMed

    Primakoff, P; Hyatt, H

    1986-09-01

    The zona-free hamster egg penetration assay (HEPA) was evaluated as a test for identifying fertilization-blocking antibodies. A monoclonal antibody, AH-20, that binds to the surface of guinea pig sperm was used to test antibody inhibition of sperm-egg fusion. AH-20 strongly inhibited guinea pig sperm fusion with zona-free hamster eggs but had no effect on guinea pig sperm fusion with zona-free guinea pig eggs. No inhibition by AH-20 was found in the homologous fusion assay over a wide range of sperm concentration, fertilization rate, and fertilization index. The results suggest that although guinea pig sperm can fuse with both hamster and guinea pig eggs, some aspect of the fusion mechanism is different in the two cases. The findings also indicate that HEPA, which is frequently used to assess the fertility potential of human sperm, can identify as blockers of sperm-egg fusion antibodies that have no effect on homologous sperm-egg fusion.

  11. Metabolic fuel homeostasis in Syrian hamsters: nycthemeral and exercise variables.

    PubMed

    Rowland, N

    1984-08-01

    The food intake and growth of male and female golden hamsters, with and without access to running wheels, were measured under several conditions. These included different seasons, photoperiods and diets. Chow-fed males in wheels invariably had slowed weight gain and showed little hyperphagia relative to sedentary controls. One group of exercising female hamsters, fed high fat diet, grew faster than sedentary controls. The food intake of both exercising and sedentary groups was distributed evenly through the day/night cycle, but the exercising animals took smaller, more frequent meals at night. The nycthemeral variations in plasma glucose, triglycerides, free fatty acids and liver glycogen were quite small in sedentary hamsters. In exercising hamsters, however, liver glycogen was elevated in the late daytime and depleted in the mid-to-late night. In vivo lipogenesis rates in white and brown adipose and liver were elevated by day in the exercising compared to sedentary hamsters, and were lower at night in both groups. It appears that in exercising hamsters, and to a lesser extent sedentary hamsters, the day phase is one of inactivity and fuel storage, and the night phase is one of exercise and fuel mobilization.

  12. Penetrating eye injuries.

    PubMed Central

    Patel, B C

    1989-01-01

    A review of all penetrating eye injuries treated at the Manchester Royal Eye Hospital over four years (1 January 1982-31 December 1985) was undertaken. A total of 202 penetrating eye injuries were seen of which 68 (34%) were in children under the age of 15 years. Airgun, dart, and knife injuries accounted for 28 (41%) of the injuries. Thirty seven patients (54%) achieved a good visual result (6/12 or better) and eight (12%) had enucleations. The period of inpatient treatment ranged from two to 18 days. From the analysis of the activities at the time of the injury, many of the injuries can be considered to be preventable. PMID:2705791

  13. Ground-penetrating rada

    NASA Astrophysics Data System (ADS)

    Thuma, W. R.

    The theory and applications of digital Ground-Penetrating Radar were discussed at a 5-day seminar held at the China University of Geosciences in Wuhan, People's Republic of China, in April. Cohosted by the Department of Applied Geophysics and Canada-China Geoscience, more than 60 senior geophysicists, engineers, technical specialists, university professors and researchers attended.Focus of the meeting was the expanded uses of the new deep-penetrating fully digital PulseEKKO, which is gaining wide acceptance around the world. Attendees showed intense interest in this new and unique technology. Applications covered were groundwater and mineral exploration; engineering, construction and toxic waste site surveying; tunnel and underground mine probing for potential geological hazards, blind ore zones, karst cavities and solution pathways; and locating buried objects such as petroleum storage tanks, unexploded bombs and archeological remains.

  14. Antibody tumor penetration

    PubMed Central

    Thurber, Greg M.; Schmidt, Michael M.; Wittrup, K. Dane

    2009-01-01

    Antibodies have proven to be effective agents in cancer imaging and therapy. One of the major challenges still facing the field is the heterogeneous distribution of these agents in tumors when administered systemically. Large regions of untargeted cells can therefore escape therapy and potentially select for more resistant cells. We present here a summary of theoretical and experimental approaches to analyze and improve antibody penetration in tumor tissue. PMID:18541331

  15. Penetrating extremity trauma.

    PubMed

    Ivatury, Rao R; Anand, Rahul; Ordonez, Carlos

    2015-06-01

    Penetrating extremity trauma (PET) usually becomes less important when present along with multiple truncal injuries. The middle eastern wars documented the terrible mortality and morbidity resulting from PET. Even in civilian trauma, PET can lead to significant morbidity and mortality. There are now well-established principles in the evaluation and management of vascular, bony, soft tissue, and neurologic lesions that will lead to a reduction of the poor outcomes. This review will summarize some of these recent concepts.

  16. Ultrastructure of immature and mature human oocytes after cryotop vitrification

    PubMed Central

    PALMERINI, Maria Grazia; ANTINORI, Monica; MAIONE, Marta; CERUSICO, Fabrizio; VERSACI, Caterina; NOTTOLA, Stefania Annarita; MACCHIARELLI, Guido; KHALILI, Mohammad Ali; ANTINORI, Severino

    2014-01-01

    In vitro maturation of vitrified immature germinal vesicle (GV) oocytes is a promising fertility preservation option. We analyzed the ultrastructure of human GV oocytes after Cryotop vitrification (GVv) and compared it with fresh GV (GVc), fresh mature metaphase II (MIIc) and Cryotop-vitrified mature (MIIv) oocytes. By phase contrast microscopy and light microscopy, the oolemmal and cytoplasmic organization of fresh and vitrified oocytes did not show significant changes. GVv oocytes showed significant ultrastructural alterations of the microvilli in 40% of the samples; small vacuoles and occasional large/isolated vacuoles were abnormally present in the ooplasm periphery of 50% of samples. The ultrastructure of nuclei and mitochondria-vesicle (MV) complexes, as well as the distribution and characteristics of cortical granules (CGs), were comparable with those of GVc oocytes. MIIv oocytes showed an abnormal ultrastructure of microvilli in 30% of the samples and isolated large vacuoles in 70% of the samples. MV complexes were normal, but mitochondria-smooth endoplasmic reticulum aggregates appeared to be of reduced size. CGs were normally located under the oolemma but presented abnormalities in distribution and matrix electron density. In conclusion, Cryotop vitrification preserved main oocyte characteristics in the GV and MII stages, even if peculiar ultrastructural alterations appeared in both stages. This study also showed that the GV stage appears more suitable for vitrification than the MII stage, as indicated by the good ultrastructural preservation of important structures that are present only in immature oocytes, like the nucleus and migrating CGs. PMID:25168087

  17. PTK2b function during fertilization of the mouse oocyte

    SciTech Connect

    Luo, Jinping; McGinnis, Lynda K.; Carlton, Carol; Beggs, Hilary E.; Kinsey, William H.

    2014-08-01

    Highlights: • PTK2b is expressed in oocytes and is activated following fertilization. • PTK2b suppression in oocytes prevents fertilization, but not parthenogenetic activation. • PTK2b suppression prevents the oocyte from fusing with or incorporating bound sperm. • PTK2b suppressed oocytes that fail to fertilize do not exhibit calcium oscillations. - Abstract: Fertilization triggers rapid changes in intracellular free calcium that serve to activate multiple signaling events critical to the initiation of successful development. Among the pathways downstream of the fertilization-induced calcium transient is the calcium-calmodulin dependent protein tyrosine kinase PTK2b or PYK2 kinase. PTK2b plays an important role in fertilization of the zebrafish oocyte and the objective of the present study was to establish whether PTK2b also functions in mammalian fertilization. PTK2b was activated during the first few hours after fertilization of the mouse oocyte during the period when anaphase resumption was underway and prior to the pronuclear stage. Suppression of PTK2b kinase activity in oocytes blocked sperm incorporation and egg activation although sperm-oocyte binding was not affected. Oocytes that failed to incorporate sperm after inhibitor treatment showed no evidence of a calcium transient and no evidence of anaphase resumption suggesting that egg activation did not occur. The results indicate that PTK2b functions during the sperm-egg fusion process or during the physical incorporation of sperm into the egg cytoplasm and is therefore critical for successful development.

  18. Distribution of protein disulfide isomerase during maturation of pig oocytes.

    PubMed

    Ohashi, Yumi; Hoshino, Yumi; Tanemura, Kentaro; Sato, Eimei

    2013-01-01

    Oocyte maturation in mammals is characterized by a dramatic reorganization of the endoplasmic reticulum (ER). In mice, the ER forms accumulations in the germinal vesicle (GV) stage and distinctive cortical clusters in metaphase II (MII) of the oocyte. Multiple evidence suggests that this ER distribution is important in preparing the oocyte for Ca(2+) oscillations, which trigger oocyte activation at fertilization. In this study, we investigated the time course and illustrated the possible functional role of ER distribution during maturation of porcine oocytes by immunostaining with protein disulfide isomerase (PDI). PDI forms clusters in the cytoplasm of oocytes. After immunostaining, PDI clusters were identified throughout the cytoplasm from the GV to metaphase I (MI) stage; however, at the MII stage, the PDI formed large clusters (1-2 µm) in the animal pole around the first polar body. PDI distribution was prevented by bacitracin, a PDI inhibitor. Our experiments indicated that, during porcine oocyte maturation, PDI undergoes a dramatic reorganization. This characteristic distribution is different from that in the mouse oocyte. Moreover, our study suggested that formation of PDI clusters in the animal pole is a specific characteristic of matured porcine oocytes. PMID:23302077

  19. High survival of mouse oocytes using an optimized vitrification protocol

    PubMed Central

    Zhou, Cheng-Jie; Wang, Dong-Hui; Niu, Xin-Xin; Kong, Xiang-Wei; Li, Yan-Jiao; Ren, Jing; Zhou, Hong-Xia; Lu, Angeleem; Zhao, Yue-Fang; Liang, Cheng-Guang

    2016-01-01

    The method of vitrification has been widely used for cryopreservation. However, the effectiveness of this method for mammalian oocytes could be improved by optimizing each step of the process. In the present study, we tested the effects of varying several key parameters to determine the most effective protocol for mouse oocyte vitrification. We found that cryoprotectant containing ethylene glycol and dimethylsulfoxide plus 20% fetal calf serum produced the highest rates of oocyte survival, fertilization, and blastocyst formation. The duration and temperature of oocyte exposure to vitrification and thawing solutions influenced survival rate. The presence of cumulus cells surrounding oocytes and the incubation of thawed oocytes in Toyoda-Yokoyama-Hosoki medium also increased oocyte survival. Open pulled straw and nylon loop methods were more effective than the mini-drop method. Finally, the combination of these improved methods resulted in better spindle morphology when compared to the unimproved methods. These results demonstrate that the outcomes of mouse oocyte vitrification can be improved by a suitable combination of cryopreservation methods, which could be applied to future clinical research with human oocytes. PMID:26781721

  20. Mars penetrator: Subsurface science mission

    NASA Technical Reports Server (NTRS)

    Lumpkin, C. K.

    1974-01-01

    A penetrator system to emplace subsurface science on the planet Mars is described. The need for subsurface science is discussed, and the technologies for achieving successful atmospheric entry, Mars penetration, and data retrieval are presented.

  1. Cryopreservation of in vitro matured oocytes after ex vivo oocyte retrieval from gynecologic cancer patients undergoing radical surgery

    PubMed Central

    Park, Chan Woo; Lee, Sun Hee; Yang, Kwang Moon; Lee, In Ho; Lim, Kyung Teak; Lee, Ki Heon

    2016-01-01

    Objective The aim of this study was to report a case series of in vitro matured (IVM) oocyte freezing in gynecologic cancer patients undergoing radical surgery under time constraints as an option for fertility preservation (FP). Methods Case series report. University-based in vitro fertilization center. Six gynecologic cancer patients who were scheduled to undergo radical surgery the next day were referred for FP. The patients had endometrial (n=2), ovarian (n=3), and double primary endometrial and ovarian (n=1) cancer. Ex vivo retrieval of immature oocytes from macroscopically normal ovarian tissue was followed by mature oocyte freezing after IVM or embryo freezing with intracytoplasmic sperm injection. Results A total of 53 oocytes were retrieved from five patients, with a mean of 10.6 oocytes per patient. After IVM, a total of 36 mature oocytes were obtained, demonstrating a 67.9% maturation rate. With regard to the ovarian cancer patients, seven IVM oocytes were frozen from patient 3, who had stage IC cancer, whereas one IVM oocyte was frozen from patient 4, who had stage IV cancer despite being of a similar age. With regard to the endometrial cancer patients, 15 IVM oocytes from patient 1 were frozen. Five embryos were frozen after the fertilization of IVM oocytes from patient 6. Conclusion Immature oocytes can be successfully retrieved ex vivo from macroscopically normal ovarian tissue before radical surgery. IVM oocyte freezing provides a possible FP option in patients with advanced-stage endometrial or ovarian cancer without the risk of cancer cell spillage or time delays. PMID:27358831

  2. A role for glucose in hypothermic hamsters

    NASA Technical Reports Server (NTRS)

    Resch, G. E.; Musacchia, X. J.

    1976-01-01

    Hypothermic hamsters at a rectal temperature of 7 C showed a fivefold increase in survival times from 20 to 100.5 hr when infused with glucose which maintained a blood level at about 45 mg/100 ml. A potential role for osmotic effects of the infusion was tested and eliminated. There was no improvement in survival of 3-O-methylglucose or dextran 40-infused animals. The fact that death eventually occurs even in the glucose-infused animal after about 4 days and that oxygen consumption undergoes a slow decrement in that period suggests that hypothermic survival is not wholly substrate limited. Radioactive tracer showed that localization of the C-14 was greatest in brain tissue and diaphragm, intermediate in heart and kidney, and lowest in skeletal muscle and liver. The significance of the label at sites important to respiration and circulation was presented.

  3. A study of the carcinogenicity of glycidol in Syrian hamsters.

    PubMed

    Lijinsky, W; Kovatch, R M

    1992-01-01

    The industrial chemical glycidol is a directly acting mutagen and a broadly acting carcinogen in rats. It was administered to Syrian golden hamsters (20 male and 20 female) by gavage of 12 mg twice a week for 60 weeks. The total dose per animal was 1.45 g or 20 mmol. Survival was not different from control hamsters treated with corn oil/ethyl acetate. Of the treated males, 9 had tumors and 13 of the treated females had tumors, some of which were adrenal cortex tumors seen in controls. More tumors were seen in the glycidol-treated hamsters than in controls, but the spleen was the only notable target organ and the number of animals with spleen hemangiosarcomas was small. Glycidol appeared to be less carcinogenic in hamsters than in rats or mice.

  4. 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

  5. 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

  6. The deep penetrating nevus.

    PubMed

    Strazzula, Lauren; Senna, Maryanne Makredes; Yasuda, Mariko; Belazarian, Leah

    2014-12-01

    The deep penetrating nevus (DPN), also known as the plexiform spindle cell nevus, is a pigmented lesion that commonly arises on the head and neck in the first few decades of life. Histopathologically, the DPN is wedge-shaped and contains melanocytes that exhibit deep infiltration into the dermis. Given these features, DPN may clinically and histopathologically mimic malignant melanoma, sparking confusion about the appropriate evaluation and management of these lesions. The goal of this review is to summarize the clinical and histopathological features of DPN and to discuss diagnostic and treatment strategies for dermatologists.

  7. Evidence for a metabolic limitation of survival in hypothermic hamsters.

    NASA Technical Reports Server (NTRS)

    Prewitt, R. L.; Anderson, G. L.; Musacchia, X. J.

    1972-01-01

    The underlying factors limiting survival in the hypothermic state are studied. Hamsters of both sexes, clipped and unclipped, were inducted into profound hypothermia by the helium cold method until they reached a temperature between 7 and 10 C. It appears that the primary cause of death is failure of respiration due to the depletion of carbohydrate energy supplies and may explain why survival time in hypothermia is shorter than the normal hibernation time of the hamster.

  8. Histaminergic regulation of seasonal metabolic rhythms in Siberian hamsters.

    PubMed

    I'anson, Helen; Jethwa, Preeti H; Warner, Amy; Ebling, Francis J P

    2011-06-01

    We investigated whether histaminergic tone contributes to the seasonal catabolic state in Siberian hamsters by determining the effect of ablation of histaminergic neurons on food intake, metabolic rate and body weight. A ribosomal toxin (saporin) conjugated to orexin-B was infused into the ventral tuberomammillary region of the hypothalamus, since most histaminergic neurons express orexin receptors. This caused not only 75-80% loss of histaminergic neurons in the posterior hypothalamus, but also some loss of other orexin-receptor expressing cells e.g. MCH neurons. In the long-day anabolic state, lesions produced a transient post-surgical decrease in body weight, but the hamsters recovered and maintained constant body weight, whereas weight gradually increased in sham-lesioned hamsters. VO(2) in the dark phase was significantly higher in the lesioned hamsters compared to shams, and locomotor activity also tended to be higher. In a second study in short days, sham-treated hamsters showed the expected seasonal decrease in body weight, but weight remained constant in the lesioned hamsters, as in the long-day study. Lesioned hamsters consumed more during the early dark phase and less during the light phase due to an increase in the frequency of meals during the dark and decreased meal size during the light, and their cumulative food intake in their home cages was greater than in the control hamsters. In summary, ablation of orexin-responsive cells in the posterior hypothalamus blocks the short-day induced decline in body weight by preventing seasonal hypophagia, evidence consistent with the hypothesis that central histaminergic mechanisms contribute to long-term regulation of body weight.

  9. Fasting-induced daily torpor in desert hamsters (Phodopus roborovskii).

    PubMed

    Chi, Qing-Sheng; Wan, Xin-Rong; Geiser, Fritz; Wang, De-Hua

    2016-09-01

    Daily torpor is frequently expressed in small rodents when facing energetically unfavorable ambient conditions. Desert hamsters (Phodopus roborovskii, ~20g) appear to be an exception as they have been described as homeothermic. However, we hypothesized that they can use torpor because we observed reversible decreases of body temperature (Tb) in fasted hamsters. To test this hypothesis we (i) randomly exposed fasted summer-acclimated hamsters to ambient temperatures (Tas) ranging from 5 to 30°C or (ii) supplied them with different rations of food at Ta 23°C. All desert hamsters showed heterothermy with the lowest mean Tb of 31.4±1.9°C (minimum, 29.0°C) and 31.8±2.0°C (minimum, 29.0°C) when fasted at Ta of 23°C and 19°C, respectively. Below Ta 19°C, the lowest Tb and metabolic rate increased and the proportion of hamsters using heterothermy declined. At Ta 5°C, nearly all hamsters remained normothermic by increasing heat production, suggesting that the heterothermy only occurs in moderately cold conditions, perhaps to avoid freezing at extremely low Tas. During heterothermy, Tbs below 31°C with metabolic rates below 25% of those during normothermia were detected in four individuals at Ta of 19°C and 23°C. Consequently, by definition, our observations confirm that fasted desert hamsters are capable of shallow daily torpor. The negative correlation between the lowest Tbs and amount of food supply shows that heterothermy was mainly triggered by food shortage. Our data indicate that summer-acclimated desert hamsters can express fasting-induced shallow daily torpor, which may be of significance for energy conservation and survival in the wild.

  10. Fasting-induced daily torpor in desert hamsters (Phodopus roborovskii).

    PubMed

    Chi, Qing-Sheng; Wan, Xin-Rong; Geiser, Fritz; Wang, De-Hua

    2016-09-01

    Daily torpor is frequently expressed in small rodents when facing energetically unfavorable ambient conditions. Desert hamsters (Phodopus roborovskii, ~20g) appear to be an exception as they have been described as homeothermic. However, we hypothesized that they can use torpor because we observed reversible decreases of body temperature (Tb) in fasted hamsters. To test this hypothesis we (i) randomly exposed fasted summer-acclimated hamsters to ambient temperatures (Tas) ranging from 5 to 30°C or (ii) supplied them with different rations of food at Ta 23°C. All desert hamsters showed heterothermy with the lowest mean Tb of 31.4±1.9°C (minimum, 29.0°C) and 31.8±2.0°C (minimum, 29.0°C) when fasted at Ta of 23°C and 19°C, respectively. Below Ta 19°C, the lowest Tb and metabolic rate increased and the proportion of hamsters using heterothermy declined. At Ta 5°C, nearly all hamsters remained normothermic by increasing heat production, suggesting that the heterothermy only occurs in moderately cold conditions, perhaps to avoid freezing at extremely low Tas. During heterothermy, Tbs below 31°C with metabolic rates below 25% of those during normothermia were detected in four individuals at Ta of 19°C and 23°C. Consequently, by definition, our observations confirm that fasted desert hamsters are capable of shallow daily torpor. The negative correlation between the lowest Tbs and amount of food supply shows that heterothermy was mainly triggered by food shortage. Our data indicate that summer-acclimated desert hamsters can express fasting-induced shallow daily torpor, which may be of significance for energy conservation and survival in the wild. PMID:27215346

  11. The effect of FF-MAS on porcine cumulus-oocyte complex maturation, fertilization and pronucleus formation in vitro.

    PubMed

    Faerge, Inger; Strejcek, Frantisek; Laurincik, Jozef; Rath, Detlef; Niemann, Heiner; Schellander, Karl; Rosenkranz, Christine; Hyttel, Poul Maddox; Grøndahl, Christian

    2006-08-01

    Follicular fluid meiosis-activating sterol (FF-MAS) has been isolated from the follicular fluid (FF) of several species including man. FF-MAS increases the quality of in vitro oocyte maturation, and thus the developmental potential of oocytes exposed to FF-MAS during in vitro maturation is improved. The aim of the present study was to investigate the effects of FF-MAS on porcine oocyte maturation and pronucleus formation in vitro. Porcine cumulus-oocyte complexes (COCs) were isolated from abattoir ovaries and in vitro matured for 48 h in NCSU 37 medium supplemented with 1 mg/l cysteine, 10 ng/ml epidermal growth factor and 50 microM 2-mercaptoethanol with or without 10% porcine follicular fluid (pFF). For the first 22 h, 1 mM db-cAMP and 10 I.E PMSG/hCG was added. The medium was supplemented with 1 microM, 3 microM, 10 microM, 30 microM or 100 microM FF-MAS dissolved in ethanol. After maturation the COCs were denuded mechanically using a fine glass pipette under constant pH and in vitro fertilized with fresh semen (5 x 10(5) spermatozoa/ml). The presumptive zygotes were evaluated 18 h after fertilization. The addition of pFF increased the monospermic as well as the polyspermic penetration of oocytes. In the absence of pFF, the addition of FF-MAS decreased the polyspermic penetration rate, whereas FF-MAS in combination with pFF decreased monospermic and increased polyspermic penetration. The degeneration rate of ova decreased in the presence of FF-MAS irrespective of the presence or absence of pFF. In the absence of pFF, FF-MAS at 3-10 microM increased the number of zygotes with advanced maternal pronuclear stages. In supraphysiological doses, i.e. 30-100 microM, FF-MAS dose-dependently and reversibly inhibited nuclear maturation in the absence of pFF.

  12. Effect of lower than expected number of oocyte on the IVF results after oocyte-pickup

    PubMed Central

    Gonca, Süheyla; Gün, Ismet; Ovayolu, Ali; Şilfeler, Dilek; Sofuoğlu, Kenan; Özdamar, Özkan; Yilmaz, Ali; Tunali, Gülden

    2014-01-01

    Objectives: To investigate whether a lower than expected number of oocyte after ≥14 mm follicle aspiration during OPU has any effect on pregnancy outcomes Methods: This is a retrospective study done between 2010 and 2013 at the IVF Unit of the Zeynep Kamil Women and Children Diseases Education and Research Hospital, dealing with the medical records of infertile patients who underwent IVF cycle and controlled ovarian stimulation with long agonist or fix antogonist protocol. The patients included into the study were those diagnosed with a primary infertility, aged between 23 and 39, at a BMI of 22-28 kg/m2 and having received the first or second IVF treatment. Male factor, presence of uterine anomaly, patients with serious endometriosis and patients with low ovarian reserve were all excluded from the study. Typically, oocyte pick-up was performed in all the patients 35.5 hours after the hCG implementation. Single or double embryo transfer was performed, where available. Patients were classified into two groups. Group 1 consisted of those with no difference between ≥14 mm aspirated follicle number and expected number of oocyte or with 1 missing number of oocyte at the most. Group 2 consisted of those with at least ≥2 missing number of oocyte between aspirated follicle number and expected number of oocyte. Statistical analysis was performed using Student’s t test for continuous variables and chi-square test for categorical variables. Additionally, a Linear regression analysis was conducted between the total number of oocyte and pregnancy. Results: In total, 387 treatment cycles were included into the study. Group 1 consisted of 134 patients and Group 2 consisted of 252 patients. Antral follicle number (12.8 ± 4.3 and 14.5 ± 4.1, P = 0.0007), hCG day E2 value (1990.7 ± 1056.4 and 2515.2 ± 1332.7, P < 0.0001) and the the number of aspirated follicle during OPU (9.1 ± 4.4 and 13.7 ± 5.5, P < 0.0001) were significantly higher in Group 2; whereas on the other

  13. Cryopreservation of embryos and oocytes: an update.

    PubMed

    Gelety, T; Surrey, E

    1993-10-01

    Widespread incorporation of human embryo cryopreservation into IVF programs may reduce the risk of multiple gestation and severe ovarian hyperstimulation syndrome as well as contributing to an overall increase in pregnancy rates. Slow cooling techniques appropriate to the stage of embryo development are most commonly employed. Growing experience with ultrarapid freezing suggests that this technique may offer similar success rates and has the advantages of simplicity and economy. The developmental stage at cryopreservation has not been conclusively shown to influence successful pregnancy outcome, although several retrospective studies suggest improved embryo survival and pregnancy rate following freezing at the pronuclear stage. Endometrial preparation using a number of different regimens before thawed-embryo transfer appears to confer no advantage over the natural cycle, but may be necessary with ovulatory dysfunction. The use of gonadotropin-releasing hormone (GnRH) analogs during ovarian stimulation for IVF has been suggested to decrease subsequent embryo freeze-thaw survival rates. However, increased pregnancy rates have been reported following transfer of previously cryopreserved embryos originating in cycles using GnRH analog (GnRHa), possibly reflecting increased numbers of embryos available for freezing. Cryopreservation of embryos originating from fertile donor oocytes has been very successful and may be used to facilitate synchronization between a donor and one or more recipients. Recent work in oocyte cryopreservation has addressed the problems of meiotic spindle disruption, the risks of aneuploidy, and decreased fertilization associated with zona hardening and polyspermy. Further refinements in technique will be required before widespread oocyte banking becomes feasible. PMID:8241436

  14. Maternal factors required for oocyte developmental competence in mice: transcriptome analysis of non-surrounded nucleolus (NSN) and surrounded nucleolus (SN) oocytes.

    PubMed

    Ma, Jun-Yu; Li, Mo; Luo, Yi-Bo; Song, Shuhui; Tian, Dongmei; Yang, Jin; Zhang, Bing; Hou, Yi; Schatten, Heide; Liu, Zhonghua; Sun, Qing-Yuan

    2013-06-15

    During mouse antral follicle development, the oocyte chromatin gradually transforms from a less condensed state with no Hoechst-positive rim surrounding the nucleolus (NSN) to a fully condensed chromatin state with a Hoechst-positive rim surrounding the nucleolus (SN). Compared with SN oocytes, NSN oocytes display a higher gene transcription activity and a lower rate of meiosis resumption (G2/M transition), and they are mostly arrested at the two-cell stage after in vitro fertilization. To explore the differences between NSN and SN oocytes, and the maternal factors required for oocyte developmental competence, we compared the whole-transcriptome profiles between NSN and SN oocytes. First, we found that the NSN and SN oocytes were different in their metabolic pathways. In the phosphatidylinositol signaling pathway, the SN oocytes tend to produce diacylglycerol, whereas the NSN oocytes tend to produce phosphatidylinositol (3,4,5)-trisphosphate. For energy production, the SN oocytes and NSN oocytes differed in the gluconeogenesis and in the synthesis processes. Second, we also found that the key genes associated with oocyte meiosis and/or preimplantation embryo development were differently expressed in the NSN and SN oocytes. Our results illustrate that during the NSN-SN transition, the oocytes change their metabolic activities and accumulate maternal factors for further oocyte maturation and post-fertilization embryo development.

  15. Regulation of tonic gonadotropin release in prepubertal female hamsters

    SciTech Connect

    Smith, S.G.; Matt, K.S.; Prestowitz, W.F.; Stetson, M.H.

    1982-04-01

    Basal serum gonadotropin levels were monitored weekly in female hamsters from birth to 10 weeks of age. Hamsters raised on three different photoperiods presented uniform pre- and postpubertal patterns of serum LH and FSH, suggesting that gonadotropin release in the young hamster occurs independently of ambient photoperiod. In all groups, serum LH levels increased gradually in animals up to 4 weeks of age, after which levels plateaued at 50--100 ng/ml. Serum FSH was markedly elevated in 2- and 3-week-old hamsters (800--1200 ng/ml), but remained at 200--400 ng/ml in all other groups. We next examined the change in the responsiveness of the pituitary to exogenous gonadotropin-releasing hormone (GnRH) challenge. Female hamsters 2 days of age failed to respond to any dose (0.025--1000 ng) of GnRH, while 10-day old females responded in typical dose-dependent fashion. GnRH-stimulated LH release first occurred in 6-day-old hamsters and was maximal by day 9, whereas FSH release first occurred on day 8 and was maximal by day 9. The prepubertal pattern of gonadotropin release can, in part, be explained on the basis of the development of pituitary GnRH sensitivity, which occurs independently of photoperiod.

  16. Regulation of hamster splenocyte reactivity to concanavalin A during pregnancy

    SciTech Connect

    Weppner, W.A.; Coggin, J.H. Jr.

    1980-08-15

    The survival to term of mammalian fetuses regardless of their expression of paternal or embryonic developmental antigens suggests that some alteration in the immune capabilities of a female occur during pregnancy. The immunocompetence of female Syrian golden hamsters during pregnancy was investigated with respect to the blastogenic response of spleen cells to the T-cell mitogen concanavalin A (Con A). The blastogenic response of spleen cells from pregnant hamsters during mid- or late gestation is 10% of that observed for spleen cells from age-matched, virgin female animals. The spleen cells from pregnant hamsters are not capable of suppressing the proliferative response of spleen cells from virgin females to Con A. However, the serum from pregnant hamsters, in comparison with serum from virgin female animals, is capable of reducing this mitogenic response. Extensive washing of the splenocytes from pregnant hamsters does reduce the degree of suppression. These results suggest that the hamster is an excellent animal model for the investigation of the mechanism(s) of immune regulation that operate during pregnancy.

  17. Weather entrainment and multispectral diel activity rhythm of desert hamsters.

    PubMed

    Wan, Xinrong; Zhang, Xinjie; Huo, Yingjun; Wang, Guiming

    2013-10-01

    The circadian rhythm of animals is an adaptation to predictable variation in environmental conditions. Multiple internal oscillators may allow animals to cope with environmental oscillations in different frequencies. Heat stress and dramatic differences between night and day temperatures are the main selective pressures of the diel activity of desert mammals, particularly small-sized rodents. We tested the hypotheses that the diel activities of desert hamsters (Phodopus roborovskii) would be entrained by ambient humidity and temperature. We predicted that increases in night temperature and humidity would improve the propensity to perform activities of the hamster. We observed hourly activities of desert hamsters under semi natural conditions for 24 consecutive hours, with seven replicates in 7 different days. We fit generalized linear mixed models to observed proportions of active hamsters, temperatures, and relative humidity. Observed diel activities of desert hamsters consisted of three harmonic oscillations in the periodicities of 24 h, 12 h, and 6 h, respectively. Furthermore, probabilities to perform activities were positively related to night temperature and humidity. Therefore, the diel activities of desert hamsters are synchronized by atmospheric humidity, temperatures, and environmental cues of ultradian fluctuations.

  18. Suppression of hamster lymphocyte reactivity to simian virus 40 tumor surface antigens by spleen cells from pregnant hamsters

    SciTech Connect

    Weppner, W.A.; Adkinson, L.R.; Coggin, J.H.Jr

    1980-09-01

    SV40-transformed tumor cells in hamsters have been found to have cell surface antigens cross-reactive with antigens temporally expressed on fetal tissues. Using a lymphocyte transformation assay, spleen cells from pregnant hamsters were found to be incapable of responding to preparations of either hamster fetal tissue or SV40-transformed cells. However, a suppressor component can be demonstrated in spleen cell populations of both primi-and multiparous hamsters during pregnancy that is capable of reducing the response of lymphocytes sensitized against SV40 tumor-associated antigens. The degree of suppression is proportional to the ratio of responder cells to spleen cells from pregnant animals. These results suggest there is a subpopulation of spleen cells involved in immunoregulation during pregnancy that has the ability to suppress the reactivity of lymphocytes sensitized against SV40-associated oncofetal antigens.

  19. Overview: Hard Rock Penetration

    SciTech Connect

    Dunn, J.C.

    1992-01-01

    The Hard Rock Penetration program is developing technology to reduce the costs of drilling and completing geothermal wells. Current projects include: lost circulation control, rock penetration mechanics, instrumentation, and industry/DOE cost shared projects of the Geothermal Drilling organization. Last year, a number of accomplishments were achieved in each of these areas. A new flow meter being developed to accurately measure drilling fluid outflow was tested extensively during Long Valley drilling. Results show that this meter is rugged, reliable, and can provide useful measurements of small differences in fluid inflow and outflow rates. By providing early indications of fluid gain or loss, improved control of blow-out and lost circulation problems during geothermal drilling can be expected. In the area of downhole tools for lost circulation control, the concept of a downhole injector for injecting a two-component, fast-setting cementitious mud was developed. DOE filed a patent application for this concept during FY 91. The design criteria for a high-temperature potassium, uranium, thorium logging tool featuring a downhole data storage computer were established, and a request for proposals was submitted to tool development companies. The fundamental theory of acoustic telemetry in drill strings was significantly advanced through field experimentation and analysis. A new understanding of energy loss mechanisms was developed.

  20. Overview: Hard Rock Penetration

    SciTech Connect

    Dunn, J.C.

    1992-08-01

    The Hard Rock Penetration program is developing technology to reduce the costs of drilling and completing geothermal wells. Current projects include: lost circulation control, rock penetration mechanics, instrumentation, and industry/DOE cost shared projects of the Geothermal Drilling organization. Last year, a number of accomplishments were achieved in each of these areas. A new flow meter being developed to accurately measure drilling fluid outflow was tested extensively during Long Valley drilling. Results show that this meter is rugged, reliable, and can provide useful measurements of small differences in fluid inflow and outflow rates. By providing early indications of fluid gain or loss, improved control of blow-out and lost circulation problems during geothermal drilling can be expected. In the area of downhole tools for lost circulation control, the concept of a downhole injector for injecting a two-component, fast-setting cementitious mud was developed. DOE filed a patent application for this concept during FY 91. The design criteria for a high-temperature potassium, uranium, thorium logging tool featuring a downhole data storage computer were established, and a request for proposals was submitted to tool development companies. The fundamental theory of acoustic telemetry in drill strings was significantly advanced through field experimentation and analysis. A new understanding of energy loss mechanisms was developed.

  1. Overview - Hard Rock Penetration

    SciTech Connect

    Dunn, James C.

    1992-03-24

    The Hard Rock Penetration program is developing technology to reduce the costs of drilling and completing geothermal wells. Current projects include: lost circulation control, rock penetration mechanics, instrumentation, and industry/DOE cost shared projects of the Geothermal Drilling Organization. Last year, a number of accomplishments were achieved in each of these areas. A new flow meter being developed to accurately measure drilling fluid outflow was tested extensively during Long Valley drilling. Results show that this meter is rugged, reliable, and can provide useful measurements of small differences in fluid inflow and outflow rates. By providing early indications of fluid gain or loss, improved control of blow-out and lost circulation problems during geothermal drilling can be expected. In the area of downhole tools for lost circulation control, the concept of a downhole injector for injecting a two-component, fast-setting cementitious mud was developed. DOE filed a patent application for this concept during FY 91. The design criteria for a high-temperature potassium, uranium, thorium logging tool featuring a downhole data storage computer were established, and a request for proposals was submitted to tool development companies. The fundamental theory of acoustic telemetry in drill strings was significantly advanced through field experimentation and analysis. A new understanding of energy loss mechanisms was developed.

  2. Monolithic ballasted penetrator

    DOEpatents

    Hickerson, Jr., James P.; Zanner, Frank J.; Baldwin, Michael D.; Maguire, Michael C.

    2001-01-01

    The present invention is a monolithic ballasted penetrator capable of delivering a working payload to a hardened target, such as reinforced concrete. The invention includes a ballast made from a dense heavy material insert and a monolithic case extending along an axis and consisting of a high-strength steel alloy. The case includes a nose end containing a hollow portion in which the ballast is nearly completely surrounded so that no movement of the ballast relative to the case is possible during impact with a hard target. The case is cast around the ballast, joining the two parts together. The ballast may contain concentric grooves or protrusions that improve joint strength between the case and ballast. The case further includes a second hollow portion; between the ballast and base, which has a payload fastened within this portion. The penetrator can be used to carry instrumentation to measure the geologic character of the earth, or properties of arctic ice, as they pass through it.

  3. Oocyte maturation and quality: role of cyclic nucleotides.

    PubMed

    Gilchrist, R B; Luciano, A M; Richani, D; Zeng, H T; Wang, X; Vos, M De; Sugimura, S; Smitz, J; Richard, F J; Thompson, J G

    2016-11-01

    The cyclic nucleotides, cAMP and cGMP, are the key molecules controlling mammalian oocyte meiosis. Their roles in oocyte biology have been at the forefront of oocyte research for decades, and many of the long-standing controversies in relation to the regulation of oocyte meiotic maturation are now resolved. It is now clear that the follicle prevents meiotic resumption through the actions of natriuretic peptides and cGMP - inhibiting the hydrolysis of intra-oocyte cAMP - and that the pre-ovulatory gonadotrophin surge reverses these processes. The gonadotrophin surge also leads to a transient spike in cAMP in the somatic compartment of the follicle. Research over the past two decades has conclusively demonstrated that this surge in cAMP is important for the subsequent developmental capacity of the oocyte. This is important, as oocyte in vitro maturation (IVM) systems practised clinically do not recapitulate this cAMP surge in vitro, possibly accounting for the lower efficiency of IVM compared with clinical IVF. This review particularly focuses on this latter aspect - the role of cAMP/cGMP in the regulation of oocyte quality. We conclude that clinical practice of IVM should reflect this new understanding of the role of cyclic nucleotides, thereby creating a new generation of ART and fertility treatment options. PMID:27422885

  4. Oxidative stress and ageing of the post-ovulatory oocyte.

    PubMed

    Lord, Tessa; Aitken, R John

    2013-12-01

    With extended periods of time following ovulation, the metaphase II stage oocyte experiences deterioration in quality referred to as post-ovulatory oocyte ageing. Post-ovulatory ageing occurs both in vivo and in vitro and has been associated with reduced fertilization rates, poor embryo quality, post-implantation errors and abnormalities in the offspring. Although the physiological consequences of post-ovulatory oocyte ageing have largely been established, the molecular mechanisms controlling this process are not well defined. This review analyses the relationships between biochemical changes exhibited by the ageing oocyte and the symptoms associated with the ageing phenotype. We also discuss molecular events that are potentially involved in orchestrating post-ovulatory ageing with a particular focus on the role of oxidative stress. We propose that oxidative stress may act as the initiator for a cascade of events that create the aged oocyte phenotype. Specifically, oxidative stress has the capacity to cause a decline in levels of critical cell cycle factors such as maturation-promoting factor, impair calcium homoeostasis, induce mitochondrial dysfunction and directly damage multiple intracellular components of the oocyte such as lipids, proteins and DNA. Finally, this review addresses current strategies for delaying post-ovulatory oocyte ageing with a particular focus on the potential use of compounds such as caffeine or selected antioxidants in the development of more refined media for the preservation of oocyte integrity during IVF procedures.

  5. The type and extent of injuries in vitrified mouse oocytes.

    PubMed

    Liang, Yang; Ning, Fang-Yong; Du, Wen-Jing; Wang, Chun-Sheng; Piao, Shan-Hua; An, Tie-Zhu

    2012-04-01

    To improve the vitrification of mouse oocytes using straws, we attempted to estimate the type and extent of injuries during vitrification with a vitrification solution EAFS10/10. Injuries in oocytes were assessed based on cellular viability, the integrity of the plasma membrane, the status of the meiotic spindle/chromosomes, and morphological appearance. For morphologically normal oocytes, the ability to be fertilized and to develop into blastocysts was examined. Morphological assessment revealed 15% of oocytes to be injured by intracellular ice formed during vitrification, and 10% by osmotic swelling during removal of the cryoprotectant. When assessed by the status of spindles/chromosomes, the most sensitive criterion, damage was found in 16% of oocytes without any treatment. This value was similar to the proportion of fresh oocytes that did not cleave after insemination (13%). On exposure to EAFS10/10, the spindles/chromosomes were affected in 33% of oocytes. The exposure reduced the rate of cleavage by 18% points and the rate of development into blastocysts by 19 points. Vitrification reduced these rates by 15% and 36% points, respectively. Although the mechanism responsible for this moderate toxic effect on developmental ability is not known, information obtained in the present study will be useful to develop a practical method for the vitrification of mouse oocytes using straws.

  6. The type and extent of injuries in vitrified mouse oocytes.

    PubMed

    Liang, Yang; Ning, Fang-Yong; Du, Wen-Jing; Wang, Chun-Sheng; Piao, Shan-Hua; An, Tie-Zhu

    2012-04-01

    To improve the vitrification of mouse oocytes using straws, we attempted to estimate the type and extent of injuries during vitrification with a vitrification solution EAFS10/10. Injuries in oocytes were assessed based on cellular viability, the integrity of the plasma membrane, the status of the meiotic spindle/chromosomes, and morphological appearance. For morphologically normal oocytes, the ability to be fertilized and to develop into blastocysts was examined. Morphological assessment revealed 15% of oocytes to be injured by intracellular ice formed during vitrification, and 10% by osmotic swelling during removal of the cryoprotectant. When assessed by the status of spindles/chromosomes, the most sensitive criterion, damage was found in 16% of oocytes without any treatment. This value was similar to the proportion of fresh oocytes that did not cleave after insemination (13%). On exposure to EAFS10/10, the spindles/chromosomes were affected in 33% of oocytes. The exposure reduced the rate of cleavage by 18% points and the rate of development into blastocysts by 19 points. Vitrification reduced these rates by 15% and 36% points, respectively. Although the mechanism responsible for this moderate toxic effect on developmental ability is not known, information obtained in the present study will be useful to develop a practical method for the vitrification of mouse oocytes using straws. PMID:22202671

  7. On-chip enucleation of an oocyte by untethered microrobots

    NASA Astrophysics Data System (ADS)

    Ichikawa, Akihiko; Sakuma, Shinya; Sugita, Masakuni; Shoda, Tatsuro; Tamakoshi, Takahiro; Akagi, Satoshi; Arai, Fumihito

    2014-09-01

    We propose a novel on-chip enucleation of an oocyte with zona pellucida by using a combination of untethered microrobots. To achieve enucleation within the closed space of a microfluidic chip, two microrobots, a microknife and a microgripper were integrated into the microfluidic chip. These microrobots were actuated by an external magnetic force produced by permanent magnets placed on the robotic stage. The tip of the microknife was designed by considering the biological geometric feature of an oocyte, i.e. the oocyte has a polar body in maturation stage II. Moreover, the microknife was fabricated by using grayscale lithography, which allows fabrication of three-dimensional microstructures. The microgripper has a gripping function that is independent of the driving mechanism. On-chip enucleation was demonstrated, and the enucleated oocytes are spherical, indicating that the cell membrane of the oocytes remained intact. To confirm successful enucleation using this method, we investigated the viability of oocytes after enucleation. The results show that the production rate, i.e. the ratio between the number of oocytes that reach the blastocyst stage and the number of bovine oocytes after nucleus transfer, is 100%. The technique will contribute to complex cell manipulation such as cell surgery in lab-on-a-chip devices.

  8. XGef Mediates Early CPEB Phosphorylation during Xenopus Oocyte Meiotic Maturation

    PubMed Central

    Martínez, Susana E.; Yuan, Lei; Lacza, Charlemagne; Ransom, Heather; Mahon, Gwendolyn M.; Whitehead, Ian P.; Hake, Laura E.

    2005-01-01

    Polyadenylation-induced translation is an important regulatory mechanism during metazoan development. During Xenopus oocyte meiotic progression, polyadenylation-induced translation is regulated by CPEB, which is activated by phosphorylation. XGef, a guanine exchange factor, is a CPEB-interacting protein involved in the early steps of progesterone-stimulated oocyte maturation. We find that XGef influences early oocyte maturation by directly influencing CPEB function. XGef and CPEB interact during oogenesis and oocyte maturation and are present in a c-mos messenger ribonucleoprotein (mRNP). Both proteins also interact directly in vitro. XGef overexpression increases the level of CPEB phosphorylated early during oocyte maturation, and this directly correlates with increased Mos protein accumulation and acceleration of meiotic resumption. To exert this effect, XGef must retain guanine exchange activity and the interaction with CPEB. Overexpression of a guanine exchange deficient version of XGef, which interacts with CPEB, does not enhance early CPEB phosphorylation. Overexpression of a version of XGef that has significantly reduced interaction with CPEB, but retains guanine exchange activity, decreases early CPEB phosphorylation and delays oocyte maturation. Injection of XGef antibodies into oocytes blocks progesterone-induced oocyte maturation and early CPEB phosphorylation. These findings indicate that XGef is involved in early CPEB activation and implicate GTPase signaling in this process. PMID:15635100

  9. Hormonal control of mammalian oocyte meiosis at diplotene stage.

    PubMed

    Zhang, Meijia; Xia, Guoliang

    2012-04-01

    Mammalian oocytes grow and undergo meiosis within ovarian follicles. Fully grown oocytes are arrested at the first meiotic prophase by a mural granulosa origin "arrester" until a surge of luteinizing hormone (LH) from the pituitary at the mid-cycle stimulates the immature oocyte to resume meiosis. Recent evidence indicates that natriuretic peptide precursor type C (NPPC) produced by mural granulosa cells stimulates the generation of cyclic guanosine 3',5'-monophosphate (cGMP) by cumulus cell natriuretic peptide receptor 2 (NPR2), which diffuses into oocyte via gap junctions and inhibits oocyte phosphodiesterase 3A (PDE3A) activity and cyclic adenosine 3',5'-monophosphate (cAMP) hydrolysis and maintains meiotic arrest with a high intraoocyte cAMP level. This cAMP is generated through the activity of the Gs G-protein by the G-protein-coupled receptor, GPR3 and GPR12, and adenylyl cyclases (ADCY) endogenous to the oocyte. Further studies suggest that endocrine hormones, such as follicle-stimulating hormone (FSH), LH, 17β-estradiol (E2) and oocyte-derived paracrine factors (ODPFs), participate in oocyte meiosis possibly by the regulation of NPPC and/or NPR2. A detailed investigation of NPPC and NPR2 expression in follicle cells will elucidate the precise molecular mechanisms of gonadotropins, and control the arrest as well as resumption of meiosis.

  10. Xenopus oocyte meiosis lacks spindle assembly checkpoint control

    PubMed Central

    Shao, Hua; Ma, Chunqi; Chen, Eric

    2013-01-01

    The spindle assembly checkpoint (SAC) functions as a surveillance mechanism to detect chromosome misalignment and to delay anaphase until the errors are corrected. The SAC is thought to control mitosis and meiosis, including meiosis in mammalian eggs. However, it remains unknown if meiosis in the eggs of nonmammalian vertebrate species is also regulated by SAC. Using a novel karyotyping technique, we demonstrate that complete disruption of spindle microtubules in Xenopus laevis oocytes did not affect the bivalent-to-dyad transition at the time oocytes are undergoing anaphase I. These oocytes also acquired the ability to respond to parthenogenetic activation, which indicates proper metaphase II arrest. Similarly, oocytes exhibiting monopolar spindles, via inhibition of aurora B or Eg5 kinesin, underwent monopolar anaphase on time and without additional intervention. Therefore, the metaphase-to-anaphase transition in frog oocytes is not regulated by SAC. PMID:23569212

  11. Penetrating abdominal trauma.

    PubMed

    Henneman, P L

    1989-08-01

    The management of patients with penetrating abdominal trauma is outlined in Figure 1. Patients with hemodynamic instability, evisceration, significant gastrointestinal bleeding, peritoneal signs, gunshot wounds with peritoneal violation, and type 2 and 3 shotgun wounds should undergo emergency laparotomy. The initial ED management of these patients includes airway management, monitoring of cardiac rhythm and vital signs, history, physical examination, and placement of intravenous lines. Blood should be obtained for initial hematocrit, type and cross-matching, electrolytes, and an alcohol level or drug screen as needed. Initial resuscitation should utilize crystalloid fluid replacement. If more than 2 liters of crystalloid are needed to stabilize an adult (less in a child), blood should be given. Group O Rh-negative packed red blood cells should be immediately available for a patient in impending arrest or massive hemorrhage. Type-specific blood should be available within 15 minutes. A patient with penetrating thoracic and high abdominal trauma should receive a portable chest x-ray, and a hemo- or pneumothorax should be treated with tube thoracostomy. An unstable patient with clinical signs consistent with a pneumothorax, however, should receive a tube thoracostomy prior to obtaining roentgenographic confirmation. If time permits, a nasogastric tube and Foley catheter should be placed, and the urine evaluated for blood (these procedures can be performed in the operating room). If kidney involvement is suspected because of hematuria or penetrating trauma in the area of a kidney or ureter in a patient requiring surgery, a single-shot IVP should be performed either in the ED or the operating room. An ECG is important in patients with possible cardiac involvement and in patients over the age of 40 going to the operating room. Tetanus status should be updated, and appropriate antibiotics covering bowel flora should be given. Operative management should rarely be delayed

  12. In Vitro Matured Oocytes Are More Susceptible than In Vivo Matured Oocytes to Mock ICSI Induced Functional and Genetic Changes

    PubMed Central

    Salian, Sujit Raj; Singh, Vikram Jeet; Kalthur, Guruprasad; Adiga, Satish Kumar

    2015-01-01

    Background Concerns regarding the safety of ICSI have been intensified recently due to increased risk of birth defects in ICSI born children. Although fertilization rate is significantly higher in ICSI cycles, studies have failed to demonstrate the benefits of ICSI in improving the pregnancy rate. Poor technical skill, and suboptimal in vitro conditions may account for the ICSI results however, there is no report on the effects of oocyte manipulations on the ICSI outcome. Objective The present study elucidates the influence of mock ICSI on the functional and genetic integrity of the mouse oocytes. Methods Reactive Oxygen Species (ROS) level, mitochondrial status, and phosphorylation of H2AX were assessed in the in vivo matured and IVM oocytes subjected to mock ICSI. Results A significant increase in ROS level was observed in both in vivo matured and IVM oocytes subjected to mock ICSI (P<0.05-0.001) whereas unique mitochondrial distribution pattern was found only in IVM oocytes (P<0.01-0.001). Importantly, differential H2AX phosphorylation was observed in both in vivo matured and IVM oocytes subjected to mock ICSI (P <0.001). Conclusion The data from this study suggests that mock ICSI can alter genetic and functional integrity in oocytes and IVM oocytes are more vulnerable to mock ICSI induced changes. PMID:25786120

  13. The beneficial effects of cumulus cells and oocyte-cumulus cell gap junctions depends on oocyte maturation and fertilization methods in mice

    PubMed Central

    Zhou, Cheng-Jie; Wu, Sha-Na; Shen, Jiang-Peng; Wang, Dong-Hui; Kong, Xiang-Wei; Lu, Angeleem; Li, Yan-Jiao; Zhou, Hong-Xia; Zhao, Yue-Fang

    2016-01-01

    Cumulus cells are a group of closely associated granulosa cells that surround and nourish oocytes. Previous studies have shown that cumulus cells contribute to oocyte maturation and fertilization through gap junction communication. However, it is not known how this gap junction signaling affects in vivo versus in vitro maturation of oocytes, and their subsequent fertilization and embryonic development following insemination. Therefore, in our study, we performed mouse oocyte maturation and insemination using in vivo- or in vitro-matured oocyte-cumulus complexes (OCCs, which retain gap junctions between the cumulus cells and the oocytes), in vitro-matured, denuded oocytes co-cultured with cumulus cells (DCs, which lack gap junctions between the cumulus cells and the oocytes), and in vitro-matured, denuded oocytes without cumulus cells (DOs). Using these models, we were able to analyze the effects of gap junction signaling on oocyte maturation, fertilization, and early embryo development. We found that gap junctions were necessary for both in vivo and in vitro oocyte maturation. In addition, for oocytes matured in vivo, the presence of cumulus cells during insemination improved fertilization and blastocyst formation, and this improvement was strengthened by gap junctions. Moreover, for oocytes matured in vitro, the presence of cumulus cells during insemination improved fertilization, but not blastocyst formation, and this improvement was independent of gap junctions. Our results demonstrate, for the first time, that the beneficial effect of gap junction signaling from cumulus cells depends on oocyte maturation and fertilization methods. PMID:26966678

  14. Wee1B depletion promotes nuclear maturation of canine oocytes.

    PubMed

    Kim, Yu-Gon; Kim, Dong-Hoon; Song, Seok-Hwan; Lee, Kyeong-Lim; Yang, Byoung-Chul; Oh, Jeong Su; Lee, Sang-Ryeul; Kong, Il-Keun

    2015-03-01

    Most mammalian oocytes are arrested at the germinal vesicle stage by activation of Wee1B. Meiotic resumption is regulated by inactivation of Wee1B and activation of cell division cycle 25B. The aim of this study was to determine whether treatment with Wee1B-targeting small interfering RNA (Wee1B-siRNA) promotes nuclear maturation of canine oocytes from germinal vesicle stage to metaphase II (MII) stage. In experiment 1, the percentage of canine oocytes that matured to MII stage was higher (P < 0.05) among oocytes cultured in vitro for 72 hours than among those cultured for 24 and 48 hours (5.4 ± 2.5% vs. 0.0 ± 0.0% and 1.4 ± 1.0%, respectively). Furthermore, the percentage of oocytes that matured to metaphase I (MI) stage was higher (P < 0.05) among oocytes cultured for 48 and 72 hours than among those cultured for 24 hours (14.9 ± 10.0% and 22.4 ± 8.1%, respectively, vs. 5.7 ± 6.0%). In experiment 2, canine oocytes were intracytoplasmically microinjected with Wee1B-siRNA (50 μM) at various culture time points (0, 24, 48, or 72 hours). The nuclear configuration of the exception of oocytes in the 72-hour group was examined after 84 hours of culture. The percentage of oocytes that matured to the MII stage was higher (P < 0.05) among those treated with Wee1B-siRNA at 0 hours than among control oocytes and those injected at 72 hours (18.0 ± 1.7% vs. 2.1 ± 2.8% and 0.0 ± 0.0%, respectively). Moreover, the percentage of oocytes that matured to the MI stage was higher (P < 0.05) among those injected at 0 hours than among control oocytes and those injected at 24 and 72 hours (45.9 ± 6.8% vs. 22.1 ± 3.5%, 22.8 ± 10.0%, and 10.0 ± 4.4%, respectively). In experiment 3, oocytes were intracytoplasmically microinjected with Wee1B-siRNA at 0 hours of IVM and cultured for 0, 24, 48, or 72 hours. Thereafter, maturation-related gene expression was analyzed by quantitative real-time polymerase chain reaction. Messenger RNA

  15. Exogenous T3 mimics long day lengths in Siberian hamsters.

    PubMed

    Freeman, David A; Teubner, Brett J W; Smith, Carlesia D; Prendergast, Brian J

    2007-06-01

    Siberian hamsters (Phodopus sungorus) exhibit seasonal cycles of reproduction driven by changes in day length. Day length is encoded endogenously by the duration of nocturnal melatonin (Mel) secretion from the pineal gland. Short-duration Mel signals stimulate reproduction and long-duration signals inhibit reproduction. The mechanism by which Mel signals are decoded at the level of neural target tissues remains uncharacterized. In Siberian hamsters, exposure to short day lengths or injections of Mel in long days results in a decrease in hypothalamic expression of type 2 iodothyronine deiodinase (Dio2) mRNA. Dio2 catalyzes the conversion of the thyroid hormone thyroxine to triiodothyronine (T3). Thus exposure to short and long day lengths should decrease and increase hypothalamic T3 concentrations, respectively. We tested the hypothesis that exogenous T3 administered to short-day hamsters would mimic exposure to long day lengths with respect to gonadal stimulation. Hamsters gestated and raised in short day lengths that exhibited photoinhibition of the testes were given daily subutaneous injections of T3 or saline vehicle for 4 wk beginning at week 12 of life. The results indicate that exogenous T3 induced gonadal growth in short-day hamsters and delayed spontaneous gonadal development by an interval equal to the number of weeks during which T3 was administered. T3 injections delayed gonadal regression if given coincident with the transfer of hamsters from long to short day lengths. These results suggest that T3 mimics long day exposure in Siberian hamsters and may serve as an intermediate step between the Mel rhythm and the reproductive response.

  16. Copulatory and agonistic behavior in Syrian hamsters following social defeat.

    PubMed

    Jeffress, Elizabeth C; Huhman, Kim L

    2013-01-01

    Syrian hamsters are highly aggressive animals that reliably defend their home territory. After social defeat, however, hamsters no longer defend their home cage but instead display submissive and defensive behavior toward an intruder, a response that we have termed conditioned defeat. Plasma testosterone is significantly reduced in Syrian hamsters following repeated defeat suggesting that social defeat might also impair copulatory behavior. The present study aimed to determine whether copulatory behavior in male Syrian hamsters is suppressed following repeated social defeats and additionally whether exposure to a hormone-primed stimulus female after social defeat reduces the behavioral response to defeat. Hamsters were paired with an aggressive opponent for one or nine defeats using a resident-intruder model, while controls were placed into the empty cage of a resident aggressor. On the day after the last treatment, half of the hamsters were paired with a receptive female for 10 min. There were no significant differences in the copulatory behavior of defeated versus non-defeated hamsters, and the opportunity to copulate had no effect on subsequent conditioned defeat testing, as defeated animals displayed significantly more submissive behavior than did non-defeated animals. The current data suggest that conditioned defeat is not necessarily a maladaptive response to social stress, at least in terms of reproductive behavior, but may instead represent a viable behavioral strategy adopted by losing animals following social defeat. Further, these data indicate that conditioned defeat is relatively persistent and stable, as the opportunity to copulate does not reduce the subsequent display of submissive behavior.

  17. Social thermoregulation and torpor in the Siberian hamster.

    PubMed

    Jefimow, Małgorzata; Głabska, Marta; Wojciechowski, Michał S

    2011-04-01

    Social thermoregulation and huddling bring about energy benefits to animals sharing a nest because of the smaller surface-to-volume ratio of a huddle and the higher local temperature in the nest. We tested whether living in groups and huddling affect daily torpor, metabolic rate and seasonal changes in the body mass of a small heterothermic rodent, the Siberian hamster (Phodopus sungorus), housed under semi-natural conditions both singly and in groups of four litter-mates. We predicted that in hamsters housed in groups: (1) synchronized torpor bouts would be longer and deeper than non-synchronized ones but shallower than in solitary hamsters, (2) seasonal variations in metabolic rate would be lower than in solitary hamsters, and (3) the winter decrease in body mass would be smaller in grouped than in singly housed hamsters. We found that group housing led to a smaller decrease in body mass in winter, and affected the length and depth of daily torpor. In group-living hamsters more than 50% of all torpor episodes were synchronized and torpid animals were often found in huddles formed of all cage-mates. The longest and deepest torpor bouts in groups were recorded when all animals in a group entered torpor simultaneously. Although the minimum body temperature during torpor was higher, torpor duration was slightly longer than in solitary hamsters. We did not record significant differences in the body mass-adjusted rate of oxygen consumption between solitary and grouped animals, either in the cold or at the lower critical temperature. We conclude that social thermoregulation enables maintenance of a larger body mass, and thus a larger body fat content, which can ensure better body condition at the beginning of the reproductive season.

  18. Projectile penetration into representative targets

    NASA Astrophysics Data System (ADS)

    Stone, George W.

    1994-10-01

    The differential equation representing the penetration of a 'hard' projectile into semi-infinite, homogeneous target materials is solved for several generic combinations of the target material/projectile characteristics. A 'hard' projectile is defined as one that does not change size or shape and does not lose mass during the penetration process. The target materials evaluated range from the structurally 'soft' materials (liquids) to structurally 'hard' materials (armor plate) with viscous and fluid dynamic drag considered. The solutions to the differential equation(s) are expanded in series form to demonstrate the underlying parameters governing projectile penetration and the way they interact to limit penetration in a given target material. It is shown that the fundamental parameter governing projectile penetration into structurally 'firm' materials is the initial kinetic energy of the projectile divided by the frontal area of the projectile and the inherent structural characteristic of the target. Experimental data on the penetration of steel spheres into ballistic gelatin and for armor piercing bullets into armor plate materials are used to verify the characteristics of the solutions to the equation of motion for the projectile and to demonstrate how penetration can vary with projectile size and target characteristics. The penetration equation for a single 'hard' target material is used to develop a solution for the penetration of multilayered 'hard' target materials.

  19. Treatment of disseminated histoplasmosis in hamsters.

    PubMed

    Finquelievich, J L; Negroni, R; Iovannitti, C; Bava, A J

    1989-02-01

    A comparative study between itraconazole, ketoconazole and amphotericin B in the treatment of experimental histoplasmosis in hamsters was carried out. Seventy five animals were inoculated intracardiacally with the yeast-phase of Histoplasma capsulatum. They were divided in 5 groups: 1) treated with itraconazole by gavage (g) at a daily dose of 16 mg/kg; 2) treated with ketoconazole by (g) at a daily dose of 80 mg/kg; 3) treated with amphotericin B intraperitoneally (i.p.) at 6 mg/kg every other day; 4) control animals receiving distilled water i.p. and 5) control animals receiving P.E.G. 200 by (g). All the treatments were started one week after the challenge inoculation and they were given for 21 days. The results were evaluated by autopsy of all the animals one week after the end of the treatments. The following determinations were taken into account: microscopic examinations of spleen, liver and lungs and cultures of the spleen with determination of colony forming units/g. All the antifungal drugs used in this study were able to cause negative microscopic examinations of the liver, spleen and lungs; but only amphotericin B produced culture negative results. Itraconazole and ketoconazole presented 66% and 86% of positive cultures respectively, nevertheless the C.F.U. were lower than those obtained in control groups. In these experimental conditions amphotericin B seems to be more active than the azolic compounds and itraconazole is slightly superior to ketoconazole at a lower dose.

  20. Proteomic analysis of Chinese hamster ovary cells.

    PubMed

    Baycin-Hizal, Deniz; Tabb, David L; Chaerkady, Raghothama; Chen, Lily; Lewis, Nathan E; Nagarajan, Harish; Sarkaria, Vishaldeep; Kumar, Amit; Wolozny, Daniel; Colao, Joe; Jacobson, Elena; Tian, Yuan; O'Meally, Robert N; Krag, Sharon S; Cole, Robert N; Palsson, Bernhard O; Zhang, Hui; Betenbaugh, Michael

    2012-11-01

    To complement the recent genomic sequencing of Chinese hamster ovary (CHO) cells, proteomic analysis was performed on CHO cells including the cellular proteome, secretome, and glycoproteome using tandem mass spectrometry (MS/MS) of multiple fractions obtained from gel electrophoresis, multidimensional liquid chromatography, and solid phase extraction of glycopeptides (SPEG). From the 120 different mass spectrometry analyses generating 682,097 MS/MS spectra, 93,548 unique peptide sequences were identified with at most 0.02 false discovery rate (FDR). A total of 6164 grouped proteins were identified from both glycoproteome and proteome analysis, representing an 8-fold increase in the number of proteins currently identified in the CHO proteome. Furthermore, this is the first proteomic study done using the CHO genome exclusively, which provides for more accurate identification of proteins. From this analysis, the CHO codon frequency was determined and found to be distinct from humans, which will facilitate expression of human proteins in CHO cells. Analysis of the combined proteomic and mRNA data sets indicated the enrichment of a number of pathways including protein processing and apoptosis but depletion of proteins involved in steroid hormone and glycosphingolipid metabolism. Five-hundred four of the detected proteins included N-acetylation modifications, and 1292 different proteins were observed to be N-glycosylated. This first large-scale proteomic analysis will enhance the knowledge base about CHO capabilities for recombinant expression and provide information useful in cell engineering efforts aimed at modifying CHO cellular functions. PMID:22971049

  1. Universal penetration test apparatus with fluid penetration sensor

    DOEpatents

    Johnson, Phillip W.; Stampfer, Joseph F.; Bradley, Orvil D.

    1999-01-01

    A universal penetration test apparatus for measuring resistance of a material to a challenge fluid. The apparatus includes a pad saturated with the challenge fluid. The apparatus includes a compression assembly for compressing the material between the pad and a compression member. The apparatus also includes a sensor mechanism for automatically detecting when the challenge fluid penetrates the material.

  2. Universal penetration test apparatus with fluid penetration sensor

    DOEpatents

    Johnson, P.W.; Stampfer, J.F.; Bradley, O.D.

    1999-02-02

    A universal penetration test apparatus is described for measuring resistance of a material to a challenge fluid. The apparatus includes a pad saturated with the challenge fluid. The apparatus includes a compression assembly for compressing the material between the pad and a compression member. The apparatus also includes a sensor mechanism for automatically detecting when the challenge fluid penetrates the material. 23 figs.

  3. Factors other than genotype account largely for the phenotypic variation of the pulmonary valve in Syrian hamsters.

    PubMed

    Fernández, M Carmen; Durán, Ana C; Fernández, Borja; Arqué, Josep M; Anderson, Robert H; Sans-Coma, Valentín

    2012-07-01

    Understanding of the aetiology of congenitally anomalous pulmonary valves remains incomplete. The aim of our study, therefore, was to elucidate the degree to which the phenotypic variation known to exist for the pulmonary valve relies on genotypic variation. Initially, we tested the hypothesis that genetically alike individuals would display similar valvar phenotypes if the phenotypic arrangement depended entirely, or almost entirely, on the genotype. Thus, we examined pulmonary valves from 982 Syrian hamsters belonging to two families subject to systematic inbreeding by crossing siblings. Their coefficient of inbreeding was 0.999 or higher, so they could be considered genetically alike. External environmental factors were standardized as much as possible. A further 97 Syrian hamsters from an outbred colony were used for comparative purposes. In both the inbred and outbred hamsters, we found valves with a purely trifoliate, or tricuspid, design, trifoliate valves with a more or less extensive fusion of the right and left leaflets, bifoliate, or bicuspid, valves with fused right and left leaflets, with or without a raphe located in the conjoined arterial sinus, and quadrifoliate, or quadricuspid, valves. The incidence of the different valvar morphological variants was similar in the outbred and inbred colonies, except for the bifoliate pulmonary valves, which were significantly more frequent in the hamsters from one of the two inbred families. Results of crosses between genetically alike hamsters revealed no significant association between the pulmonary valvar phenotypes as seen in the parents and their offspring. The incidence of bifoliate pulmonary valves, nonetheless, was higher than statistically expected in the offspring of crosses where at least one of the parents possessed a pulmonary valve with two leaflets. Our observations are consistent with the notion that the basic design of the pulmonary valve, in terms of whether it possesses three or two leaflets

  4. Progesterone influences cytoplasmic maturation in porcine oocytes developing in vitro

    PubMed Central

    Jin, Yong-Xun; Kwon, Jeong-Woo

    2016-01-01

    Progesterone (P4), an ovarian steroid hormone, is an important regulator of female reproduction. In this study, we explored the influence of progesterone on porcine oocyte nuclear maturation and cytoplasmic maturation and development in vitro. We found that the presence of P4 during oocyte maturation did not inhibit polar body extrusions but significantly increased glutathione and decreased reactive oxygen species (ROS) levels relative to that in control groups. The incidence of parthenogenetically activated oocytes that could develop to the blastocyst stage was higher (p < 0.05) when oocytes were exposed to P4 as compared to that in the controls. Cell numbers were increased in the P4-treated groups. Further, the P4-specific inhibitor mifepristone (RU486) prevented porcine oocyte maturation, as represented by the reduced incidence (p < 0.05) of oocyte first polar body extrusions. RU486 affected maturation promoting factor (MPF) activity and maternal mRNA polyadenylation status. In general, these data show that P4 influences the cytoplasmic maturation of porcine oocytes, at least partially, by decreasing their polyadenylation, thereby altering maternal gene expression.

  5. Mitochondrial dynamics and their intracellular traffic in porcine oocytes.

    PubMed

    Yamochi, T; Hashimoto, S; Amo, A; Goto, H; Yamanaka, M; Inoue, M; Nakaoka, Y; Morimoto, Y

    2016-08-01

    Meiotic maturation of oocytes requires a variety of ATP-dependent reactions, such as germinal vesicle breakdown, spindle formation, and rearrangement of plasma membrane structure, which is required for fertilization. Mitochondria are accordingly expected be localized to subcellular sites of energy utilization. Although microtubule-dependent cellular traffic for mitochondria has been studied extensively in cultured neuronal (and some other somatic) cells, the molecular mechanism of their dynamics in mammalian oocytes at different stages of maturation remains obscure. The present work describes dynamic aspects of mitochondria in porcine oocytes at the germinal vesicle stage. After incubation of oocytes with MitoTracker Orange followed by centrifugation, mitochondria-enriched ooplasm was obtained using a glass needle and transferred into a recipient oocyte. The intracellular distribution of the fluorescent mitochondria was then observed over time using a laser scanning confocal microscopy equipped with an incubator. Kinetic analysis revealed that fluorescent mitochondria moved from central to subcortical areas of oocytes and were dispersed along plasma membranes. Such movement of mitochondria was inhibited by either cytochalasin B or cytochalasin D but not by colcemid, suggesting the involvement of microfilaments. This method of visualizing mitochondrial dynamics in live cells permits study of the pathophysiology of cytoskeleton-dependent intracellular traffic of mitochondria and associated energy metabolism during meiotic maturation of oocytes.

  6. Impact of stress on oocyte quality and reproductive outcome.

    PubMed

    Prasad, Shilpa; Tiwari, Meenakshi; Pandey, Ashutosh N; Shrivastav, Tulsidas G; Chaube, Shail K

    2016-01-01

    Stress is an important factor that affects physical and mental status of a healthy person disturbing homeostasis of the body. Changes in the lifestyle are one of the major causes that lead to psychological stress. Psychological stress could impact the biology of female reproduction by targeting at the level of ovary, follicle and oocyte. The increased level of stress hormone such as cortisol reduces estradiol production possibly by affecting the granulosa cell functions within the follicle, which results deterioration in oocyte quality. Adaptation of lifestyle behaviours may generate reactive oxygen species (ROS) in the ovary, which further affects female reproduction. Balance between level of ROS and antioxidants within the ovary are important for maintenance of female reproductive health. Physiological level of ROS modulates oocyte functions, while its accumulation leads to oxidative stress (OS). OS triggers apoptosis in majority of germ cells within the ovary and even in ovulated oocytes. Although both mitochondria- as well as death-receptor pathways are involved in oocyte apoptosis, OS-induced mitochondria-mediated pathway plays a major role in the elimination of majority of germ cells from ovary. OS in the follicular fluid deteriorates oocyte quality and reduces reproductive outcome. On the other hand, antioxidants reduce ROS levels and protect against OS-mediated germ cell apoptosis and thereby depletion of germ cells from the ovary. Indeed, OS is one of the major factors that has a direct negative impact on oocyte quality and limits female reproductive outcome in several mammalian species including human. PMID:27026099

  7. Progesterone influences cytoplasmic maturation in porcine oocytes developing in vitro

    PubMed Central

    Jin, Yong-Xun; Kwon, Jeong-Woo

    2016-01-01

    Progesterone (P4), an ovarian steroid hormone, is an important regulator of female reproduction. In this study, we explored the influence of progesterone on porcine oocyte nuclear maturation and cytoplasmic maturation and development in vitro. We found that the presence of P4 during oocyte maturation did not inhibit polar body extrusions but significantly increased glutathione and decreased reactive oxygen species (ROS) levels relative to that in control groups. The incidence of parthenogenetically activated oocytes that could develop to the blastocyst stage was higher (p < 0.05) when oocytes were exposed to P4 as compared to that in the controls. Cell numbers were increased in the P4-treated groups. Further, the P4-specific inhibitor mifepristone (RU486) prevented porcine oocyte maturation, as represented by the reduced incidence (p < 0.05) of oocyte first polar body extrusions. RU486 affected maturation promoting factor (MPF) activity and maternal mRNA polyadenylation status. In general, these data show that P4 influences the cytoplasmic maturation of porcine oocytes, at least partially, by decreasing their polyadenylation, thereby altering maternal gene expression. PMID:27672508

  8. Oocyte transfer and gamete intrafallopian transfer in the mare.

    PubMed

    Carnevale, E M

    2004-07-01

    Methods for the collection and transfer of equine oocytes have been developed, and uses of these techniques have resulted in new clinical and research possibilities. Because oocyte transfer avoids reproductive problems associated with the oviduct, uterus, and cervix, pregnancies can be produced from many mares that cannot carry a pregnancy or produce embryos. Oocytes for clinical transfers are usually collected from preovulatory follicles and cultured for a short interval or transferred directly into a recipient's oviduct. For oocyte transfer, the recipient is inseminated within the uterus. A large number (1 x 10(9) to 2 x 10(9)) of motile sperms are preferred for inseminations. In contrast, sperm and oocyte are transferred into the oviduct during gamete intrafallopian transfer (GIFT). Therefore, a lower number (1 x 10(5) to 2 x 10(5)) of sperm can be used. Potentially, GIFT could be used in situations where sperm numbers are limited. Use of oocyte transfer and GIFT in clinical and research settings will aid us in understanding the interactions between oocyte, sperm, and oviduct in the equine. PMID:15271484

  9. Strategies to support human oocyte development in vitro.

    PubMed

    Telfer, Evelyn E; McLaughlin, Marie

    2012-01-01

    Many young cancer patients are now being given the option to store ovarian cortical biopsies before undergoing potentially damaging chemo- or radiotherapy. This tissue mainly contains large numbers of immature primordial follicles. Currently the only option to restore fertility using this tissue is by transplantation which may not be a viable option for all patients. Greater options to realise the potential of this tissue to restore fertility could be achieved by the development of in vitro systems that support oocyte development. The ability to develop human oocytes from the most immature stages of follicles (primordial) through to maturation and fertilisation in vitro would revolutionise fertility preservation practice. This has been achieved in mouse where in vitro grown (IVG) oocytes from primordial follicles have resulted in the production of live offspring. However, developing IVG systems to support complete development of human oocytes has been more difficult because of differences in scale of timing and size. Our lab has been working on a multi-step culture system to support growth and development of bo-vine and human oocytes from primordial through to fully grown, using fresh and cryopreserved ovarian cortical tissue. This review outlines the approaches being taken to obtain complete in vitro development of human oocytes and strategies for assessing the health and viability of IVG oocytes.

  10. Comparison of the cryo-tolerance of vitrified gorgonian oocytes.

    PubMed

    Tsai, Sujune; Yang, Vivian; Lin, Chiahsin

    2016-01-01

    Coral reefs have been declining considerably in recent years because of changes to the environment and climate. The cryopreservation of coral gametes is an essential alternative method that yields immense success in preserving corals. This study focuses on developing vitrification techniques for Junceella fragilis and Ellisella robusta oocytes, and presents a comparison on the cryotolerance of their vitrified oocytes. The results revealed that these coral oocytes could be preserved for a longer period in equilibration solution 2 and vitrification solution (VS) 2 at 5 °C than at 26 °C. Oocyte viability decreased significantly when VS2 was used for >4 min at 26 °C compared with the control. Cryoprotectant tolerance was higher in E. robusta oocytes than in J. fragilis oocytes. However, E. robusta was determined to be more cryo-tolerant, with differences attributed to their habitats, thus making E. robusta is likely a superior candidate species for further study. The results of this study on the effects of coral cryopreservation provide a foundation for developing protocols further for the cryopreservation of the oocytes of gorgonian corals. PMID:26984101

  11. Progesterone influences cytoplasmic maturation in porcine oocytes developing in vitro.

    PubMed

    Yuan, Bao; Liang, Shuang; Jin, Yong-Xun; Kwon, Jeong-Woo; Zhang, Jia-Bao; Kim, Nam-Hyung

    2016-01-01

    Progesterone (P4), an ovarian steroid hormone, is an important regulator of female reproduction. In this study, we explored the influence of progesterone on porcine oocyte nuclear maturation and cytoplasmic maturation and development in vitro. We found that the presence of P4 during oocyte maturation did not inhibit polar body extrusions but significantly increased glutathione and decreased reactive oxygen species (ROS) levels relative to that in control groups. The incidence of parthenogenetically activated oocytes that could develop to the blastocyst stage was higher (p < 0.05) when oocytes were exposed to P4 as compared to that in the controls. Cell numbers were increased in the P4-treated groups. Further, the P4-specific inhibitor mifepristone (RU486) prevented porcine oocyte maturation, as represented by the reduced incidence (p < 0.05) of oocyte first polar body extrusions. RU486 affected maturation promoting factor (MPF) activity and maternal mRNA polyadenylation status. In general, these data show that P4 influences the cytoplasmic maturation of porcine oocytes, at least partially, by decreasing their polyadenylation, thereby altering maternal gene expression. PMID:27672508

  12. Comparison of the cryo-tolerance of vitrified gorgonian oocytes

    PubMed Central

    Tsai, Sujune; Yang, Vivian; Lin, Chiahsin

    2016-01-01

    Coral reefs have been declining considerably in recent years because of changes to the environment and climate. The cryopreservation of coral gametes is an essential alternative method that yields immense success in preserving corals. This study focuses on developing vitrification techniques for Junceella fragilis and Ellisella robusta oocytes, and presents a comparison on the cryotolerance of their vitrified oocytes. The results revealed that these coral oocytes could be preserved for a longer period in equilibration solution 2 and vitrification solution (VS) 2 at 5 °C than at 26 °C. Oocyte viability decreased significantly when VS2 was used for >4 min at 26 °C compared with the control. Cryoprotectant tolerance was higher in E. robusta oocytes than in J. fragilis oocytes. However, E. robusta was determined to be more cryo-tolerant, with differences attributed to their habitats, thus making E. robusta is likely a superior candidate species for further study. The results of this study on the effects of coral cryopreservation provide a foundation for developing protocols further for the cryopreservation of the oocytes of gorgonian corals. PMID:26984101

  13. Anonymous and non-anonymous oocyte donation preliminary results.

    PubMed

    Junca, A M; Cohen, J; Mandelbaum, J; Alnot, M O; Debache, C; Plachot, M; Pez, J P

    1988-01-01

    During the past year, we have developed an oocyte donation programme in 10 patients with complete absence of endogenous ovarian function (premature ovarian failure in seven cases, castration in two cases and Turner's syndrome in one case). In cases of anonymous donation, donors were volunteers devoid of any major genetical risk who were included in our IVF programme and who consented to donate one oocyte when at least seven oocytes were recovered, and two oocytes when at least 11 oocytes were recovered, to a recipient couple. As far as possible, morphological characteristics of both couples were paired. In cases of non-anonymous donation, donors were 'affective' donors, having at least one child. The resulting embryos after IVF of donated oocytes were either replaced directly in recipient women which required synchronization of the donor's and recipient's cycles, or cryopreserved and then thawed, usually at day 16 of recipient's artificial cycle, i.e. 2 days after introduction of the progestational compound. On the 10 patients entering this oocyte donation programme (20 cycles), 13 transfers were carried out resulting in four clinical pregnancies in three patients with premature ovarian failure and one with Turner's syndrome (20% pregnancy per cycle and 31% per transfer). Despite the small numbers, these good results prompted us to develop this protocol. PMID:3350933

  14. Sequence-specific minor groove binding ligands as potential regulators of gene expression in Xenopus laevis oocytes.

    PubMed

    Belikov, S V; Grokhovsky, S L; Isaguliants, M G; Surovaya, A N; Gursky, G V

    2005-10-01

    The mouse mammary tumor virus (MMTV) promoter is induced by glucocorticoid hormone. A robust hormone- and receptor-dependent gene activation could be reproduced in Xenopus laevis oocytes. The homogeneous response in this system allowed a detailed analysis of the DNA-protein interactions following hormone activation. The strategy of artificial regulating of gene activity by sequence-specific minor groove binding ligands is very attractive. We have synthesized and studied the interaction with DNA of bis-linked netropsin derivatives in which two monomers are attached via short linkers in head-to-head and tail-to-tail manners. We have found that cis-diammine-platinum bridged bis-netropsin added to Xenopus oocytes media penetrates cellular and nuclear membrane and binds selectively to the MMTV promoter at the DNA segment that partly overlaps with the site recognized by glucocorticoid receptor. DNase I footprinting studies demonstrate that there are more stronger binding sites for cis-diammine-platinum bridged bis-netropsin on the naked MMTV DNA which are found to be inaccessible for its binding in oocytes. PMID:16060693

  15. Mutations in TUBB8 cause human oocyte meiotic arrest

    PubMed Central

    Feng, Ruizhi; Sang, Qing; Kuang, Yanping; Sun, Xiaoxi; Yan, Zheng; Zhang, Shaozhen; Shi, Juanzi; Tian, Guoling; Luchniak, Anna; Fukuda, Yusuke; Li, Bin; Yu, Min; Chen, Junling; Xu, Yao; Guo, Luo; Qu, Ronggui; Wang, Xueqian; Sun, Zhaogui; Liu, Miao; Shi, Huijuan; Wang, Hongyan; Feng, Yi; Shao, Ruijin; Chai, Renjie; Li, Qiaoli; Xing, Qinghe; Zhang, Rui; Nogales, Eva; Jin, Li; He, Lin; Gupta, Mohan L.; Cowan, Nicholas J.; Wang, Lei

    2016-01-01

    Background Successful human reproduction depends on the fusion of a mature oocyte with a sperm cell to form a fertilized egg. The genetic events that lead to human oocyte maturation arrest are unknown. Methods We recruited a rare four-generation family with female infertility as a consequence of oocyte meiosis I arrest. We applied whole-exome and direct Sanger sequencing to an additional 23 patients following identification of mutations in a candidate gene, TUBB8. Expression of TUBB8 and all other β-tubulin isotypes was measured in human oocytes, early embryos, sperm cells and several somatic tissues by qRT-PCR. The effect of the TUBB8 mutations was assessed on α/β tubulin heterodimer assembly in vitro, on microtubule architecture in HeLa cells, on microtubule dynamics in yeast cells, and on spindle assembly in mouse and human oocytes via microinjection of the corresponding cRNAs. Results We identified seven mutations in the primate-specific gene TUBB8 that are responsible for human oocyte meiosis I arrest in seven families. TUBB8 expression is unique to oocytes and the early embryo, where this gene accounts for almost all of the expressed β-tubulin. The mutations affect the chaperone-dependent folding and assembly of the α/β-tubulin heterodimer, induce microtubule chaos upon expression in cultured cells, alter microtubule dynamics in vivo, and cause catastrophic spindle assembly defects and maturation arrest upon expression in mouse and human oocytes. Conclusions TUBB8 mutations function via dominant negative effects that massively disrupt proper microtubule behavior. TUBB8 is a key gene involved in human oocyte meiotic spindle assembly and maturation. PMID:26789871

  16. Mutations in TUBB8 and Human Oocyte Meiotic Arrest.

    PubMed

    Feng, Ruizhi; Sang, Qing; Kuang, Yanping; Sun, Xiaoxi; Yan, Zheng; Zhang, Shaozhen; Shi, Juanzi; Tian, Guoling; Luchniak, Anna; Fukuda, Yusuke; Li, Bin; Yu, Min; Chen, Junling; Xu, Yao; Guo, Luo; Qu, Ronggui; Wang, Xueqian; Sun, Zhaogui; Liu, Miao; Shi, Huijuan; Wang, Hongyan; Feng, Yi; Shao, Ruijin; Chai, Renjie; Li, Qiaoli; Xing, Qinghe; Zhang, Rui; Nogales, Eva; Jin, Li; He, Lin; Gupta, Mohan L; Cowan, Nicholas J; Wang, Lei

    2016-01-21

    Background Human reproduction depends on the fusion of a mature oocyte with a sperm cell to form a fertilized egg. The genetic events that lead to the arrest of human oocyte maturation are unknown. Methods We sequenced the exomes of five members of a four-generation family, three of whom had infertility due to oocyte meiosis I arrest. We performed Sanger sequencing of a candidate gene, TUBB8, in DNA samples from these members, additional family members, and members of 23 other affected families. The expression of TUBB8 and all other β-tubulin isotypes was assessed in human oocytes, early embryos, sperm cells, and several somatic tissues by means of a quantitative reverse-transcriptase-polymerase-chain-reaction assay. We evaluated the effect of the TUBB8 mutations on the assembly of the heterodimer consisting of one α-tubulin polypeptide and one β-tubulin polypeptide (α/β-tubulin heterodimer) in vitro, on microtubule architecture in HeLa cells, on microtubule dynamics in yeast cells, and on spindle assembly in mouse and human oocytes. Results We identified seven mutations in the primate-specific gene TUBB8 that were responsible for oocyte meiosis I arrest in 7 of the 24 families. TUBB8 expression is unique to oocytes and the early embryo, in which this gene accounts for almost all the expressed β-tubulin. The mutations affect chaperone-dependent folding and assembly of the α/β-tubulin heterodimer, disrupt microtubule behavior on expression in cultured cells, alter microtubule dynamics in vivo, and cause catastrophic spindle-assembly defects and maturation arrest on expression in mouse and human oocytes. Conclusions TUBB8 mutations have dominant-negative effects that disrupt microtubule behavior and oocyte meiotic spindle assembly and maturation, causing female infertility. (Funded by the National Basic Research Program of China and others.). PMID:26789871

  17. MicroRNA Expression during Bovine Oocyte Maturation and Fertilization.

    PubMed

    Gilchrist, Graham C; Tscherner, Allison; Nalpathamkalam, Thomas; Merico, Daniele; LaMarre, Jonathan

    2016-01-01

    Successful fertilization and subsequent embryo development rely on complex molecular processes starting with the development of oocyte competence through maturation. MicroRNAs (miRNAs) are small non-coding RNA molecules that function as gene regulators in many biological systems, including the oocyte and embryo. In order to further explore the roles of miRNAs in oocyte maturation, we employed small RNA sequencing as a screening tool to identify and characterize miRNA populations present in pools of bovine germinal vesicle (GV) oocytes, metaphase II (MII) oocytes, and presumptive zygotes (PZ). Each stage contained a defined miRNA population, some of which showed stable expression while others showed progressive changes between stages that were subsequently confirmed by quantitative reverse transcription polymerase chain reaction (RT-PCR). Bta-miR-155, bta-miR-222, bta-miR-21, bta-let-7d, bta-let-7i, and bta-miR-190a were among the statistically significant differentially expressed miRNAs (p < 0.05). To determine whether changes in specific primary miRNA (pri-miRNA) transcripts were responsible for the observed miRNA changes, we evaluated pri-miR-155, -222 and let-7d expression. Pri-miR-155 and -222 were not detected in GV oocytes but pri-miR-155 was present in MII oocytes, indicating transcription during maturation. In contrast, levels of pri-let-7d decreased during maturation, suggesting that the observed increase in let-7d expression was likely due to processing of the primary transcript. This study demonstrates that both dynamic and stable populations of miRNAs are present in bovine oocytes and zygotes and extend previous studies supporting the importance of the small RNA landscape in the maturing bovine oocyte and early embryo. PMID:26999121

  18. MicroRNA Expression during Bovine Oocyte Maturation and Fertilization

    PubMed Central

    Gilchrist, Graham C.; Tscherner, Allison; Nalpathamkalam, Thomas; Merico, Daniele; LaMarre, Jonathan

    2016-01-01

    Successful fertilization and subsequent embryo development rely on complex molecular processes starting with the development of oocyte competence through maturation. MicroRNAs (miRNAs) are small non-coding RNA molecules that function as gene regulators in many biological systems, including the oocyte and embryo. In order to further explore the roles of miRNAs in oocyte maturation, we employed small RNA sequencing as a screening tool to identify and characterize miRNA populations present in pools of bovine germinal vesicle (GV) oocytes, metaphase II (MII) oocytes, and presumptive zygotes (PZ). Each stage contained a defined miRNA population, some of which showed stable expression while others showed progressive changes between stages that were subsequently confirmed by quantitative reverse transcription polymerase chain reaction (RT-PCR). Bta-miR-155, bta-miR-222, bta-miR-21, bta-let-7d, bta-let-7i, and bta-miR-190a were among the statistically significant differentially expressed miRNAs (p < 0.05). To determine whether changes in specific primary miRNA (pri-miRNA) transcripts were responsible for the observed miRNA changes, we evaluated pri-miR-155, -222 and let-7d expression. Pri-miR-155 and -222 were not detected in GV oocytes but pri-miR-155 was present in MII oocytes, indicating transcription during maturation. In contrast, levels of pri-let-7d decreased during maturation, suggesting that the observed increase in let-7d expression was likely due to processing of the primary transcript. This study demonstrates that both dynamic and stable populations of miRNAs are present in bovine oocytes and zygotes and extend previous studies supporting the importance of the small RNA landscape in the maturing bovine oocyte and early embryo. PMID:26999121

  19. Pineal melatonin synthesis in Syrian hamsters: A summary

    NASA Astrophysics Data System (ADS)

    Rollag, M. D.

    1982-12-01

    During the past decade there has been ample documentation of the proposition that the pineal gland mediates photoperiodic influences upon reproductive behavior of hamsters. It is commonly hypothesized that the pineal gland expresses its activity by transformation of photoperiodic information into an hormonal output, that hormone being melatonin. If this hypothesis is correct, there must be some essential diffrence in melatonin's output when hamsters are exposed to different photoperiodic environments. The experiments summarized in this communication analyze pineal melatonin contents in Syrian hamsters maintained in a variety of photoperiodic conditions during different physiological states. The results demonstrate that adult hamsters have a daily surge in pineal melatonin content throughout their lifetime when exposed to simulated annual photoperiodic cycles. There is some fluctuation in the amount of pineal melatonin produced during different physiological states and photoperiodic environments, but these fluctuations seem small when compared to those normally found for other regulatory hormones. When hamsters are exposed to different photoperiodic regimens, the daily melatonin surge maintains a relatively constant phase relationship with respect to the onset of daily activity. There is a concomitant change in its phase relationship with respect to light-dark transitions.

  20. Isolation, antimicrobial activities, and primary structures of hamster neutrophil defensins.

    PubMed Central

    Mak, P; Wójcik, K; Thogersen, I B; Dubin, A

    1996-01-01

    Hamster (Mesocricetus auratus) neutrophil granules contain at least four microbicidal peptides belonging to the defensin family. These compounds were purified from granule acid extracts by reverse-phase chromatography and termed HaNP-1 to -4 (hamster neutrophil peptide). HaNP-1 and HaNP-3 revealed the most bactericidal activity, with a 50% inhibitory concentration of 0.3 to 0.8 microg/ml for Staphylococcus aureus and Streptococcus pyogenes strains. The HaNP-4 was always isolated in concentrations exceeding about 10 times the concentrations of other hamster peptides, but its antibacterial activity as well as that of HaNP-2 was relatively lower, probably as a result of conserved Arg residue substitutions. Other microorganisms were also tested, and generally, hamster defensins exhibited less potency against gram-negative bacteria. The amino acid sequence of hamster defensins showed a high percentage of identity to the sequence of mouse enteric defensins, reaching about 60% identical residues in the case of HaNP-3 and cryptdin 3. PMID:8890190

  1. Interval timer control of puberty in photoinhibited Siberian hamsters.

    PubMed

    Park, Jin Ho; Kauffman, Alexander S; Paul, Matthew J; Butler, Matthew P; Beery, Annaliese K; Costantini, Ruth M; Zucker, Irving

    2006-10-01

    Puberty, which is markedly delayed in male Siberian hamsters (Phodopus sungorus) born into short day lengths, is controlled by an interval timer regulated by the duration of nocturnal melatonin secretion. Properties of the interval timer were assessed by perturbing normal patterns of melatonin secretion in males gestated and maintained thereafter in 1 of 2 short day lengths, 10 h light/day (10 L) or 12L. Melatonin secretion of short-day hamsters was suppressed by constant light treatment or modified by daily injection of propranolol to mimic nocturnal melatonin durations typical of long-day hamsters. Constant light treatment during weeks 3 to 5 induced early incomplete gonadal growth in 12L but not 10 L hamsters but did not affect late onset of gonadal development indicative of puberty in either photoperiod. Propranolol treatment during postnatal weeks 3 to 5 induced transient growth of the testes and ultimately delayed the timing of puberty by 3 weeks. Similar treatments between weeks 5 and 7 or on alternate weeks for 24 weeks did not affect the interval timer. The first 2 weeks after weaning may constitute a critical period during which the interval timer is highly responsive to photoperiod. Alternatively, the hamsters' photoperiodic history rather than age or developmental stage may be the critical variable. The interpolation of long-day melatonin signals at the time of weaning does not appear to reset the interval timer to its zero position but may reduce timer responsiveness to long-day melatonin signals several weeks later.

  2. Environmental induction of photononresponsiveness in the Siberian hamster, Phodopus sungorus.

    PubMed

    Gorman, M R; Zucker, I

    1997-03-01

    In seasonally breeding rodent species, a fraction of the population is unresponsive to short day lengths (DL) and remains reproductively competent during winter. We previously observed that incidence of nonresponsiveness to short days was affected by photoperiodic history. Here we tested whether exposure to long DL (18 h light/day; 18L) renders animals unresponsive to short DL (10L). Hamsters, maintained from birth in 10L, were transferred at week 6 to 18L or 14L. Ten weeks later (week 16), groups were transferred to 10L for 10 wk. All hamsters maintained in short DL from birth had undeveloped testes at week 6. At week 26, however, 92% of hamsters previously kept in 18L failed to undergo complete gonadal regression in 10L, compared with only 10% of hamsters previously in 14L. Entrainment of locomotor activity in 10L in nonresponsive hamsters resembled that typically observed under long DL. Exposure to 18L may induce nonresponsiveness by altering interactions of component circadian oscillators that mediate gonadal regression in short DL.

  3. The Syrian hamster model of hantavirus pulmonary syndrome

    PubMed Central

    Safronetz, David; Ebihara, Hideki; Feldmann, Heinz; Hooper, Jay W.

    2012-01-01

    Hantavirus pulmonary syndrome (HPS) is a relatively rare, but frequently fatal disease associated with New World hantaviruses, most commonly Sin Nombre and Andes viruses in North and South America, respectively. It is characterized by fever and the sudden, rapid onset of severe respiratory distress and cardiogenic shock, which can be fatal in up to 50% of cases. Currently there are no approved antiviral therapies or vaccines for the treatment or prevention of HPS. A major obstacle in the development of effective medical countermeasures against highly pathogenic agents like the hantaviruses is recapitulating the human disease as closely as possible in an appropriate and reliable animal model. To date, the only animal model that resembles HPS in humans is the Syrian hamster model. Following infection with Andes virus, hamsters develop HPS-like disease which faithfully mimics the human condition with respect to incubation period and pathophysiology of disease. Perhaps most importantly, the sudden and rapid onset of severe respiratory distress observed in humans also occurs in hamsters. The last several years has seen an increase in studies utilizing the Andes virus hamster model which have provided unique insight into HPS pathogenesis as well as potential therapeutic and vaccine strategies to treat and prevent HPS. The purpose of this article is to review the current understanding of HPS disease progression in Syrian hamsters and discuss the suitability of utilizing this model to evaluate potential medical countermeasures against HPS. PMID:22705798

  4. Hamster and murine models of severe destructive Lyme arthritis.

    PubMed

    Munson, Erik; Nardelli, Dean T; Du Chateau, Brian K; Callister, Steven M; Schell, Ronald F

    2012-01-01

    Arthritis is a frequent complication of infection in humans with Borrelia burgdorferi. Weeks to months following the onset of Lyme borreliosis, a histopathological reaction characteristic of synovitis including bone, joint, muscle, or tendon pain may occur. A subpopulation of patients may progress to a chronic, debilitating arthritis months to years after infection which has been classified as severe destructive Lyme arthritis. This arthritis involves focal bone erosion and destruction of articular cartilage. Hamsters and mice are animal models that have been utilized to study articular manifestations of Lyme borreliosis. Infection of immunocompetent LSH hamsters or C3H mice results in a transient synovitis. However, severe destructive Lyme arthritis can be induced by infecting irradiated hamsters or mice and immunocompetent Borrelia-vaccinated hamsters, mice, and interferon-gamma- (IFN-γ-) deficient mice with viable B. burgdorferi. The hamster model of severe destructive Lyme arthritis facilitates easy assessment of Lyme borreliosis vaccine preparations for deleterious effects while murine models of severe destructive Lyme arthritis allow for investigation of mechanisms of immunopathology.

  5. The hamster flank organ model: Is it relevant to man

    SciTech Connect

    Franz, T.J.; Lehman, P.A.; Pochi, P.; Odland, G.F.; Olerud, J. )

    1989-10-01

    The critical role that androgens play in the etiology of acne has led to a search for topically active antiandrogens and the frequent use of the flank organ of the golden Syrian hamster as an animal model. 17-alpha-propyltestosterone (17-PT) has been identified as having potent antiandrogenic activity in the hamster model, and this report describes its clinical evaluation. Two double-blind placebo controlled studies comparing 4% 17-PT in 80% alcohol versus vehicle alone were conducted. One study examined 17-PT sebosuppressive activity in 20 subjects. The second study examined its efficacy in 44 subjects having mild to moderate acne. A third study measured in vitro percutaneous absorption of 17-PT through hamster flank and monkey skin, and human face skin in-vivo, using radioactive drug. 17-PT was found to be ineffective in reducing either the sebum excretion rate or the number of inflammatory acne lesions. Failure of 17-PT to show clinical activity was not a result of poor percutaneous absorption. Total absorption in man was 7.7% of the dose and only 1.0% in the hamster. The sebaceous gland of hamster flank organ is apparently more sensitive to antiandrogens than the human sebaceous gland.

  6. Maturation, fertilization, and development of marmoset monkey oocytes in vitro.

    PubMed

    Gilchrist, R B; Nayudu, P L; Hodges, J K

    1997-01-01

    This study was conducted to investigate 1) the capacity of in vitro-matured (IVM) marmoset oocytes to be fertilized and to support embryonic development in vitro and 2) oocyte meiotic maturation in relation to in vivo FSH administration, follicle size, and oocyte-cumulus cell status. Pairs of ovaries were collected on Day 4 of the follicular phase from adult females receiving either 1) human FSH (3 IU; n = 5) or 2) control (saline; n = 5) daily for 4 days. Antral follicles were excised from ovaries and separated into classes according to size: class 1 (660-840 microm), class 2 (> 840-1000 microm), class 3 (> 1000-1400 microm), and class 4 (> 1400 microm). A total of 823 partially naked and cumulus-enclosed oocytes (CEOs) were released from follicles and cultured in vitro. Cumulus cells remaining after 22 h were removed, metaphase II (MII) oocytes were inseminated with epididymal sperm, and resulting embryos were cultured until developmental arrest. Fluorescence microscopy was used to assess oocyte meiotic and embryo developmental progression. Oocyte germinal vesicle breakdown (GVB)- and MII-competencies increased significantly with follicular size (p < 0.01 and p < 0.0001, respectively), although they were independent of oocyte-cumulus cell associations. After 24 and 32 h in vitro, 69% and 93%, respectively, of CEOs with MII competence had completed meiotic maturation, and the rate of nuclear maturation increased progressively with follicle size (p < 0.01) and with the association of cumulus cells (p < 0.01). In vivo FSH priming slightly improved oocyte GVB- and MII-competencies (p < 0.01 and p < 0.05, respectively) and decreased the time required to achieve MII (p < 0.01). IVM oocytes from all follicle sizes fertilized (78-92%) in vitro, with 27% developing to morula- and 4% to blastocyst-stage embryos. This study demonstrates for the first time that IVM New World primate oocytes are able to support advanced preimplantation embryonic development in vitro. Oocyte

  7. Expressing and Characterizing Mechanosensitive Channels in Xenopus Oocytes

    PubMed Central

    Maksaev, Grigory; Haswell, Elizabeth S.

    2015-01-01

    The oocytes of the African clawed frog (Xenopus laevis) comprise one of the most widely used membrane protein expression systems. While frequently used for studies of transporters and ion channels, the application of this system to the study of mechanosensitive ion channels has been overlooked, perhaps due to a relative abundance of native expression systems. Recent advances, however, have illustrated the advantages of the oocyte system for studying plant and bacterial mechanosensitive channels. Here we describe in detail the methods used for heterologous expression and characterization of bacterial and plant mechanosensitive channels in Xenopus oocytes. PMID:25981775

  8. Rapamycin Rescues the Poor Developmental Capacity of Aged Porcine Oocytes

    PubMed Central

    Lee, Seung Eun; Kim, Eun Young; Choi, Hyun Yong; Moon, Jeremiah Jiman; Park, Min Jee; Lee, Jun Beom; Jeong, Chang Jin; Park, Se Pill

    2014-01-01

    Unfertilized oocytes age inevitably after ovulation, which limits their fertilizable life span and embryonic development. Rapamycin affects mammalian target of rapamycin (mTOR) expression and cytoskeleton reorganization during oocyte meiotic maturation. The goal of this study was to examine the effects of rapamycin treatment on aged porcine oocytes and their in vitro development. Rapamycin treatment of aged oocytes for 24 h (68 h in vitro maturation [IVM]; 44 h+10 μM rapamycin/24 h, 47.52±5.68) or control oocytes (44 h IVM; 42.14±4.40) significantly increased the development rate and total cell number compared with untreated aged oocytes (68 h IVM, 22.04±5.68) (p<0.05). Rapamycin treatment of aged IVM oocytes for 24 h also rescued aberrant spindle organization and chromosomal misalignment, blocked the decrease in the level of phosphorylated-p44/42 mitogen-activated protein kinase (MAPK), and increased the mRNA expression of cytoplasmic maturation factor genes (MOS, BMP15, GDF9, and CCNB1) compared with untreated, 24 h-aged IVM oocytes (p<0.05). Furthermore, rapamycin treatment of aged oocytes decreased reactive oxygen species (ROS) activity and DNA fragmentation (p<0.05), and downregulated the mRNA expression of mTOR compared with control or untreated aged oocytes. By contrast, rapamycin treatment of aged oocytes increased mitochondrial localization (p<0.05) and upregulated the mRNA expression of autophagy (BECN1, ATG7, MAP1LC3B, ATG12, GABARAP, and GABARAPL1), anti-apoptosis (BCL2L1 and BIRC5; p<0.05), and development (NANOG and SOX2; p<0.05) genes, but it did not affect the mRNA expression of pro-apoptosis genes (FAS and CASP3) compared with the control. This study demonstrates that rapamycin treatment can rescue the poor developmental capacity of aged porcine oocytes. PMID:25049998

  9. Donor motivations, associated risks and ethical considerations of oocyte donation.

    PubMed

    Boutelle, Amy L

    2014-01-01

    Three decades after the first reported successful cases, oocyte donation continues to grow in popularity and regard as an established method to aid women in achieving their reproductive goals. As a result of the increased demand for donated oocytes, many young women in the U.S. volunteer to undergo complex medical procedures to donate their oocytes in return for financial compensation. To best care for these women before, during and after donation, it is important to explore donor characteristics and motivations, discuss the safety of the donation procedure and examine the ethical issues related to this process.

  10. A modified swim-up method reduces polyspermy during in vitro fertilization of porcine oocytes.

    PubMed

    Park, Chi-Hun; Lee, Sang-Goo; Choi, Don-Ho; Lee, Chang-Kyu

    2009-10-01

    The general method of porcine in vitro fertilization (IVF), involving the co-culture of both gametes in a medium drop, is thought to be the main reason for the high incidence of polyspermy. The aim of this study was to reduce the polyspermic fertilization of porcine embryos during IVF by the modified swim-up method, based on general sperm swim-up technique. Within this design, a 70 microm pore sized cell strainer was used to separate the sperm pellet placed at the bottom of a tube from the mature oocytes placed within the upper region. The separation of gametes using this permeable barrier was to ensure that only motile sperm gained access to the oocytes. It was found that the rate of polyspermy was significantly lowered for the sperm preparations from three boar breeds in modified swim-up method when compared with that of the general microdrop method (p<0.05). However, the penetration rates were found to be similar in both methods for two boar breeds. The average occurrence of blastocysts with more total cell number was higher in the modified swim-up method, while no significant difference in blastocyst rates between the two IVF methods was observed. The frequency of normal diploid embryos was also significantly higher in the modified swim-up method and polyploidy was more frequently observed in microdrop method (p<0.05). Our results demonstrated that the modified swim-up IVF method could reduce polyspermic penetration, and consequently produce better quality and karyotypically normal embryos in porcine IVF.

  11. Top Sounder Ice Penetration

    NASA Astrophysics Data System (ADS)

    Porter, D. L.; Goemmer, S. A.; Sweeney, J. H.

    2014-12-01

    Ice draft measurements are made as part of normal operations for all US Navy submarines operating in the Arctic Ocean. The submarine ice draft data are unique in providing high resolution measurements over long transects of the ice covered ocean. The data has been used to document a multidecadal drop in ice thickness, and for validating and improving numerical sea-ice models. A submarine upward-looking sonar draft measurement is made by a sonar transducer mounted in the sail or deck of the submarine. An acoustic beam is transmitted upward through the water column, reflecting off the bottom of the sea ice and returning to the transducer. Ice thickness is estimated as the difference between the ship's depth (measured by pressure) and the acoustic range to the bottom of the ice estimated from the travel time of the sonar pulse. Digital recording systems can provide the return off the water-ice interface as well as returns that have penetrated the ice. Typically, only the first return from the ice hull is analyzed. Information regarding ice flow interstitial layers provides ice age information and may possibly be derived with the entire return signal. The approach being investigated is similar to that used in measuring bottom sediment layers and will involve measuring the echo level from the first interface, solving the reflection loss from that transmission, and employing reflection loss versus impedance mismatch to ascertain ice structure information.

  12. Electromagnetic Field Penetration Studies

    NASA Technical Reports Server (NTRS)

    Deshpande, M.D.

    2000-01-01

    A numerical method is presented to determine electromagnetic shielding effectiveness of rectangular enclosure with apertures on its wall used for input and output connections, control panels, visual-access windows, ventilation panels, etc. Expressing EM fields in terms of cavity Green's function inside the enclosure and the free space Green's function outside the enclosure, integral equations with aperture tangential electric fields as unknown variables are obtained by enforcing the continuity of tangential electric and magnetic fields across the apertures. Using the Method of Moments, the integral equations are solved for unknown aperture fields. From these aperture fields, the EM field inside a rectangular enclosure due to external electromagnetic sources are determined. Numerical results on electric field shielding of a rectangular cavity with a thin rectangular slot obtained using the present method are compared with the results obtained using simple transmission line technique for code validation. The present technique is applied to determine field penetration inside a Boeing-757 by approximating its passenger cabin as a rectangular cavity filled with a homogeneous medium and its passenger windows by rectangular apertures. Preliminary results for, two windows, one on each side of fuselage were considered. Numerical results for Boeing-757 at frequencies 26 MHz, 171-175 MHz, and 428-432 MHz are presented.

  13. 9 CFR 3.36 - Primary enclosures used to transport live guinea pigs and hamsters.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... live guinea pigs and hamsters. 3.36 Section 3.36 Animals and Animal Products ANIMAL AND PLANT HEALTH..., Care, Treatment, and Transportation of Guinea Pigs and Hamsters Transportation Standards § 3.36 Primary enclosures used to transport live guinea pigs and hamsters. No person subject to the Animal...

  14. 9 CFR 3.36 - Primary enclosures used to transport live guinea pigs and hamsters.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... live guinea pigs and hamsters. 3.36 Section 3.36 Animals and Animal Products ANIMAL AND PLANT HEALTH..., Care, Treatment, and Transportation of Guinea Pigs and Hamsters Transportation Standards § 3.36 Primary enclosures used to transport live guinea pigs and hamsters. No person subject to the Animal...

  15. 9 CFR 3.36 - Primary enclosures used to transport live guinea pigs and hamsters.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... live guinea pigs and hamsters. 3.36 Section 3.36 Animals and Animal Products ANIMAL AND PLANT HEALTH..., Care, Treatment, and Transportation of Guinea Pigs and Hamsters Transportation Standards § 3.36 Primary enclosures used to transport live guinea pigs and hamsters. No person subject to the Animal...

  16. Penetration seals for TFTR shielding

    SciTech Connect

    Hondorp, H.L.

    1980-12-01

    The penetrations of the shielding provided for TFTR are required to be sealed to avoid radiation streaming. This report provides a discussion of the properties required for these penetration seals. Several alternate designs are discussed and evaluated and designs recommended for specific applications.

  17. An Earth Penetrating Modeling Assessment

    SciTech Connect

    Stokes, E; Yarrington, P; Glenn, L

    2005-06-21

    Documentation of a study to assess the capability of computer codes to predict lateral loads on earth penetrating projectiles under conditions of non-normal impact. Calculations simulated a set of small scale penetration tests into concrete targets with oblique faces at angles of 15 and 30 degrees to the line-of-flight. Predictive codes used by the various calculational teams cover a wide range of modeling approaches from approximate techniques, such as cavity expansion, to numerical methods, such as finite element codes. The modeling assessment was performed under the auspices of the Phenomenology Integrated Product Team (PIPT) for the Robust Nuclear Earth Penetrator Program (RNEP). Funding for the penetration experiments and modeling was provided by multiple earth penetrator programs.

  18. Static penetration resistance of soils

    NASA Technical Reports Server (NTRS)

    Durgunoglu, H. T.; Mitchell, J. K.

    1973-01-01

    Model test results were used to define the failure mechanism associated with the static penetration resistance of cohesionless and low-cohesion soils. Knowledge of this mechanism has permitted the development of a new analytical method for calculating the ultimate penetration resistance which explicitly accounts for penetrometer base apex angle and roughness, soil friction angle, and the ratio of penetration depth to base width. Curves relating the bearing capacity factors to the soil friction angle are presented for failure in general shear. Strength parameters and penetrometer interaction properties of a fine sand were determined and used as the basis for prediction of the penetration resistance encountered by wedge, cone, and flat-ended penetrometers of different surface roughness using the proposed analytical method. Because of the close agreement between predicted values and values measured in laboratory tests, it appears possible to deduce in-situ soil strength parameters and their variation with depth from the results of static penetration tests.

  19. FOXO3a is involved in the apoptosis of naked oocytes and oocytes of primordial follicles from neonatal rat ovaries.

    PubMed

    Liu, Hong; Luo, Li-Li; Qian, Yuan-Shu; Fu, Yu-Cai; Sui, Xu-Xia; Geng, Yi-Jie; Huang, Da-Na; Gao, Shi-Tong; Zhang, Ren-Li

    2009-04-17

    Inhibition of the forkhead transcription factor, FOXO3a, can promote the transition from primordial to primary follicle and subsequent follicle development in mammalian ovaries. Stem cell factor (SCF) initiates anti-apoptotic signaling from its membrane receptor, c-kit, to Bcl-2 family members through PI3K/AKT in oocytes of primordial follicles. However, whether FOXO3a mediates the apoptosis of naked oocytes and oocytes of primordial follicles remains unknown. In the present study, oocytes from nests and primordial follicles from neonatal rat ovaries were cultured, and oocyte apoptosis was examined using the TUNEL technique. The pro-apoptotic action of FOXO3a and the potential signal transduction pathways were investigated using RT-PCR, Western blot, and immunocytochemistry. Culturing oocytes in the presence of SCF did not affect the level of total FOXO3a protein, but rapidly elevated the level of phosphorylated FOXO3a (indicating functional suppression). As phosphorylated FOXO3a increased, oocyte apoptosis was inhibited. The specific PI3K/Akt inhibitor, LY 294002, abolished the phosphorylation of FOXO3a and the anti-apoptotic action of SCF. SCF down-regulated the expression of p27KIP1 and pro-apoptotic factors such as Bim, Bad, and Bax, and this activity was reversed by LY 294002. SCF up-regulated the expression of MnSOD, which was also inhibited by LY 294002. However, SCF had no effect on Bcl-2 protein. These results suggest that FOXO3a is involved in oocyte apoptosis in the neonatal rat ovary, and the SCF-PI3K/Akt-FOXO3a signaling pathway mediates oocyte apoptosis and primordial follicle formation. PMID:19258007

  20. Torpor shortens the period of Siberian hamster circadian rhythms.

    PubMed

    Thomas, E M; Jewett, M E; Zucker, I

    1993-10-01

    We investigated the influence of ambient and body temperature (Ta and Tb) on circadian rhythms of gonadectomized male Siberian hamsters. Animals that entered torpor (Tb < 30 degrees C) had significantly shorter circadian periods (tau s) than did nontorpid hamsters at a Ta of 13 degrees C (24.17 +/- 0.05 vs. 24.33 +/- 0.04 h). The tau s of homeothermic hamsters were not affected by Ta change. Short-term decreases in Tb, rather than changes in Ta, appear to affect tau. Access to activity wheels inhibited expression of torpor in short daylengths and was associated with significant increases in body mass. Running wheel activity can mask or block specific short-day responses.

  1. A Syrian golden hamster model recapitulating ebola hemorrhagic fever.

    PubMed

    Ebihara, Hideki; Zivcec, Marko; Gardner, Donald; Falzarano, Darryl; LaCasse, Rachel; Rosenke, Rebecca; Long, Dan; Haddock, Elaine; Fischer, Elizabeth; Kawaoka, Yoshihiro; Feldmann, Heinz

    2013-01-15

    Ebola hemorrhagic fever (EHF) is a severe viral infection for which no effective treatment or vaccine is currently available. While the nonhuman primate (NHP) model is used for final evaluation of experimental vaccines and therapeutic efficacy, rodent models have been widely used in ebolavirus research because of their convenience. However, the validity of rodent models has been questioned given their low predictive value for efficacy testing of vaccines and therapeutics, a result of the inconsistent manifestation of coagulopathy seen in EHF. Here, we describe a lethal Syrian hamster model of EHF using mouse-adapted Ebola virus. Infected hamsters displayed most clinical hallmarks of EHF, including severe coagulopathy and uncontrolled host immune responses. Thus, the hamster seems to be superior to the existing rodent models, offering a better tool for understanding the critical processes in pathogenesis and providing a new model for evaluating prophylactic and postexposure interventions prior to testing in NHPs.

  2. Oocyte Degeneration Associated with Follicle Cells in Female Mactra chinensis (Bivalvia: Mactridae)

    PubMed Central

    Kim, Sung Han; Chung, Ee-Yung; Lee, Ki-Young

    2014-01-01

    Ultrastructural studies of oocyte degeneration in the oocyte, and the functions of follicle cells during oocyte degeneration are described to clarify the reproductive mechanism on oocyte degeneration of Mactra chinensis using cytological methods. Commonly, the follicle cells are attached to the oocyte. Follicle cells play an important role in oocyte degeneration. In particular, the functions of follicle cells during oocyte degeneration are associated with phagocytosis and the intracellular digestion of products. In this study, morphologically similar degenerated phagosomes (various lysosomes), which were observed in the degenerated oocytes, appeared in the follicle cells. After the spawning of the oocytes, the follicle cells were involved in oocyte degeneration through phagocytosis by phagolysosomes. Therefore, it can be assumed that follicle cells reabsorb phagosomes from degenerated oocytes. In this study, the presence of lipid granules, which occurred from degenerating yolk granules, gradually increased in degenerating oocytes. The function of follicle cells can accumulate reserves of lipid granules and glycogen in the cytoplasm, which can be employed by the vitellogenic oocyte. Based on observations of follicle cells attached to degenerating oocytes after spawning, the follicle cells of this species are involved in the lysosomal induction of oocyte degeneration for the reabsorption of phagosomes (phagolysosomes) in the cytoplasm for nutrient storage, as seen in other bivalves. PMID:25949203

  3. Phenotypes of Aging Postovulatory Oocytes After Somatic Cell Nuclear Transfer in Mice.

    PubMed

    Lee, Ah Reum; Shimoike, Takashi; Wakayama, Teruhiko; Kishigami, Satoshi

    2016-06-01

    Oocytes rapidly lose their developmental potential after ovulation, termed postovulatory oocyte aging, and often exhibit characteristic phenotypes, such as cytofragmentation, abnormal spindle shapes, and chromosome misalignments. Here, we reconstructed mouse oocytes using somatic cell nuclear transfer (SCNT) to reveal the effect of somatic cell-derived nuclei on oocyte physiology during aging. Normal oocytes started undergoing cytofragmentation 24 hours after oocyte collection; however, this occurred earlier in SCNT oocytes and was more severe at 48 hours, suggesting that the transferred somatic cell nuclei affected oocyte physiology. We found no difference in the status of acetylated α-tubulin (Ac-Tub) and α-tubulin (Tub) between normal and SCNT aging oocytes, but unlike normal oocytes, aging SCNT oocytes did not have astral microtubules. Interestingly, aging SCNT oocytes displayed more severely scattered chromosomes or irregularly shaped spindles. Observations of the microfilaments showed that, in normal oocytes, there was a clear actin ring beneath the plasma membrane and condensed microfilaments around the spindle (the actin cap) at 0 hours, and the actin filaments started degenerating at 1 hour, becoming completely disrupted and distributed to the cytoplasm at 24 hours. By contrast, in SCNT oocytes, an actin cap formed around the transplanted nuclei within 1 hour of SCNT, which was still present at 24 hours. Thus, SCNT oocytes age in a similar but distinct way, suggesting that they not only contain nuclei with abnormal epigenetics but are also physiologically different. PMID:27253626

  4. Homeostatic regulation of sleep in arrhythmic Siberian hamsters.

    PubMed

    Larkin, Jennie E; Yokogawa, Tohei; Heller, H Craig; Franken, Paul; Ruby, Norman F

    2004-07-01

    Sleep is regulated by independent yet interacting circadian and homeostatic processes. The present study used a novel approach to study sleep homeostasis in the absence of circadian influences by exposing Siberian hamsters to a simple phase delay of the photocycle to make them arrhythmic. Because these hamsters lacked any circadian organization, their sleep homeostasis could be studied in the absence of circadian interactions. Control animals retained circadian rhythmicity after the phase shift and re-entrained to the phase-shifted photocycle. These animals displayed robust daily sleep-wake rhythms with consolidated sleep during the light phase beginning about 1 h after light onset. This marked sleep-wake pattern was circadian in that it persisted in constant darkness. The distribution of sleep in the arrhythmic hamsters over 24 h was similar to that in the light phase of rhythmic animals. Therefore, daily sleep amounts were higher in arrhythmic animals compared with rhythmic ones. During 2- and 6-h sleep deprivations (SD), it was more difficult to keep arrhythmic hamsters awake than it was for rhythmic hamsters. Because the arrhythmic animals obtained more non-rapid eye movement sleep (NREMS) during the SD, they showed a diminished compensatory response in NREMS EEG slow-wave activity during recovery sleep. When amounts of sleep during the SD were taken into account, there were no differences in sleep homeostasis between experimental and control hamsters. Thus loss of circadian control did not alter the homeostatic response to SD. This supports the view that circadian and homeostatic influences on sleep regulation are independent processes.

  5. The thalamic intergeniculate leaflet modulates photoperiod responsiveness in Siberian hamsters.

    PubMed

    Freeman, David A; Dhandapani, Krishnan M; Goldman, Bruce D

    2004-11-26

    Siberian hamsters are seasonal breeders that use changes in day length to synchronize their reproductive effort with those times of the year most favorable for successful reproduction. The ability of Siberian hamsters to measure and respond to changes in day length depends upon accurate photoentrainment of the circadian clock in the suprachiasmatic nucleus (SCN) of the hypothalamus. Two pathways have been characterized through which entraining stimuli reach the SCN: the retinohypothalamic tract (RHT), which transmits light information from the retinae, and the geniculohypothalamic tract (GHT) from the intergeniculate leaflet of the thalamus (IGL), which is involved in transmitting both photic and nonphotic cues. Ablating the IGL/GHT results in only modest alterations in entrainment to static day lengths and fails to interfere with seasonal responses induced by transfer from static long day to static short day lengths. Because several studies suggest that the IGL may be involved in tracking the time of dusk and dawn, we sought to determine whether an intact IGL is necessary for hamsters to respond to a simulated natural photoperiod (SNP) in which the time of dusk and dawn gradually changes in a pattern approximating the rate of change in day length that occurs during autumn at the latitude this species inhabits in nature. The results indicate that neurochemical lesions of the IGL alter both the pattern of circadian entrainment and photoperiodic responsiveness of Siberian hamsters to an SNP. Both intact and IGL-lesioned hamsters exhibited testicular regression in shortening day lengths, but only IGL-intact hamsters exhibited seasonal pelage molt.

  6. Foodborne transmission of nipah virus in Syrian hamsters.

    PubMed

    de Wit, Emmie; Prescott, Joseph; Falzarano, Darryl; Bushmaker, Trenton; Scott, Dana; Feldmann, Heinz; Munster, Vincent J

    2014-03-01

    Since 2001, outbreaks of Nipah virus have occurred almost every year in Bangladesh with high case-fatality rates. Epidemiological data suggest that in Bangladesh, Nipah virus is transmitted from the natural reservoir, fruit bats, to humans via consumption of date palm sap contaminated by bats, with subsequent human-to-human transmission. To experimentally investigate this epidemiological association between drinking of date palm sap and human cases of Nipah virus infection, we determined the viability of Nipah virus (strain Bangladesh/200401066) in artificial palm sap. At 22°C virus titers remained stable for at least 7 days, thus potentially allowing food-borne transmission. Next, we modeled food-borne Nipah virus infection by supplying Syrian hamsters with artificial palm sap containing Nipah virus. Drinking of 5×10⁸ TCID₅₀ of Nipah virus resulted in neurological disease in 5 out of 8 hamsters, indicating that food-borne transmission of Nipah virus can indeed occur. In comparison, intranasal (i.n.) inoculation with the same dose of Nipah virus resulted in lethal respiratory disease in all animals. In animals infected with Nipah virus via drinking, virus was detected in respiratory tissues rather than in the intestinal tract. Using fluorescently labeled Nipah virus particles, we showed that during drinking, a substantial amount of virus is deposited in the lungs, explaining the replication of Nipah virus in the respiratory tract of these hamsters. Besides the ability of Nipah virus to infect hamsters via the drinking route, Syrian hamsters infected via that route transmitted the virus through direct contact with naïve hamsters in 2 out of 24 transmission pairs. Although these findings do not directly prove that date palm sap contaminated with Nipah virus by bats is the origin of Nipah virus outbreaks in Bangladesh, they provide the first experimental support for this hypothesis. Understanding the Nipah virus transmission cycle is essential for preventing

  7. Maternal Photoperiodic History Affects Offspring Development in Syrian Hamsters

    PubMed Central

    Beery, Annaliese K.; Paul, Matthew J.; Routman, David M.; Zucker, Irving

    2009-01-01

    During the first 7 weeks of postnatal life, short day lengths inhibit the onset of puberty in many photoperiodic rodents, but not in Syrian hamsters. In this species, timing of puberty and fecundity are independent of the early postnatal photoperiod. Gestational day length affects postnatal reproductive development in several rodents; its role in Syrian hamsters has not been assessed. We tested the hypothesis that cumulative effects of pre- and postnatal short day lengths would restrain gonadal development in male Syrian hamsters. Males with prenatal short day exposure were generated by dams transferred to short day lengths 6 weeks, 3 weeks, and 0 weeks prior to mating. Additional groups were gestated in long day lengths and transferred to short days at birth, at 4 weeks of age, or not transferred (control hamsters). In pups of dams exposed to short day treatment throughout gestation, decreased testis growth was apparent by 3 weeks and persisted through 9 weeks of age, at which time maximum testis size was attained. A subset of males (14%), whose dams had been in short days for 3 to 6 weeks prior to mating displayed pronounced delays in testicular development, similar to those of other photoperiodic rodents. This treatment also increased the percentage of male offspring that underwent little or no gonadal regression postnatally (39%). By 19 weeks of age, males housed in short days completed spontaneous gonadal development. After prolonged long day treatment to break refractoriness, hamsters that initially were classified as nonregressors underwent testicular regression in response to a 2nd sequence of short day lengths. The combined action of prenatal and early postnatal short day lengths diminishes testicular growth of prepubertal Syrian hamsters no later than the 3rd week of postnatal life, albeit to a lesser extent than in other photoperiodic rodents. PMID:18838610

  8. Ultrastructural organization of the hamster renal pelvis.

    PubMed

    Lacy, E R; Schmidt-Nielsen, B

    1979-08-01

    The renal pelvis of the hamster has been studied by light microscopy (epoxy resin sections), transmission electron microscopy, and morphometric analysis of electron micrographs. Three morphologically distinct epithelia line the pelvis, and each covers a different zone of the kidney. A thin epithelium covering the outer medulla (OM) consists of two cell types: (1) granular cells are most numerous and have apically positioned granules which stain intensely with toluidine blue, are membrane-bound, and contain a fine particulate matter that stains light grey to black in electron micrographs. (2) Basal cells do not have granules, are confined to the basal lamina region, and do not reach the mucosal epithelial surface. The inner medulla (IM) is covered by a pelvic epithelium morphologically similar to collecting duct epithelium of IM. Some cells in this portion of the pelvic epithelium (IM) stain intensely dark with toluidine blue, osmium tetroxide, lead, and uranyl acetate. Transitional epithelium, which separates cortex (C) from pelvic urine, has an asymmetric luminal plasma membrane and discoid vesicles, each of which is similar to those previously observed in mammalian ureter and urinary bladder epithelia. Based on morphological comparisons with other epithelia, the IM and OM pelvic epithelia would appear permeable to solutes and/or water, while the transitional epithelium covering the C appears relatively impermeable. It would also appear that the exchange of solutes and water between pelvic urine and OM would involve capillaries, primarily, since morphometric analysis showed that both fenestrated and continuous capillaries of the OM were extremely abundant (greater than 60% of OM pelvic surface area) just under the thin pelvic epithelium.

  9. Deposition of salicylic acid into hamster sebaceous.

    PubMed

    Motwani, M R; Rhein, L D; Zatz, J L

    2004-01-01

    In an earlier paper, we identified vehicles that are miscible with sebum, using differential scanning calorimetry (DSC). In this paper, the potential of these vehicles to deliver salicylic acid (SA) into the sebum-filled follicles of hamster ears is examined. The main objective of this study is to correlate the melting transitions of a model sebum with the follicular delivery of SA, using two different types of vehicles (fatty and polar). Generally, the fatty vehicles show higher deposition than the polar vehicles. Follicular delivery of salicylic acid correlates well with its solubility in the respective vehicles. This extent of deposition also shows a relationship with the effect of the vehicle on thermal behavior of the model sebum. The nature of the relationship depends on the vehicle (polar or fatty) tested. We conclude that DSC could be used to identify appropriate vehicles for drugs whose follicular delivery depends on solubility. The results also suggest that delivery into the sebaceous glands occurs by two different mechanisms, depending upon the polarity of the vehicle and the physicochemical properties of the drug. The results of these experiments are further extended to investigate follicular delivery of SA from two different types of oil-in-water emulsion formulations. From these studies we conclude that either increasing the volume of the oil phase or changing the emulsion to a water-in-oil emulsion would increase follicular deposition. Our research highlights the role of sebum, its compatibility with drug molecules, and vehicle selection in the transport of drugs into the follicles. The overall results of these experiments provide a reasonable understanding of the mechanisms underlying the transport of drugs to, and subsequently through, the sebaceous follicle.

  10. Active diffusion positions the nucleus in mouse oocytes.

    PubMed

    Almonacid, Maria; Ahmed, Wylie W; Bussonnier, Matthias; Mailly, Philippe; Betz, Timo; Voituriez, Raphaël; Gov, Nir S; Verlhac, Marie-Hélène

    2015-04-01

    In somatic cells, the position of the cell centroid is dictated by the centrosome. The centrosome is instrumental in nucleus positioning, the two structures being physically connected. Mouse oocytes have no centrosomes, yet harbour centrally located nuclei. We demonstrate how oocytes define their geometric centre in the absence of centrosomes. Using live imaging of oocytes, knockout for the formin 2 actin nucleator, with off-centred nuclei, together with optical trapping and modelling, we discover an unprecedented mode of nucleus positioning. We document how active diffusion of actin-coated vesicles, driven by myosin Vb, generates a pressure gradient and a propulsion force sufficient to move the oocyte nucleus. It promotes fluidization of the cytoplasm, contributing to nucleus directional movement towards the centre. Our results highlight the potential of active diffusion, a prominent source of intracellular transport, able to move large organelles such as nuclei, providing in vivo evidence of its biological function.

  11. Recent advances in oocyte and ovarian tissue cryopreservation and transplantation

    PubMed Central

    Rodriguez-Wallberg, Kenny A.; Oktay, Kutluk

    2012-01-01

    Options for preserving fertility in women include well-established methods such as fertility-sparing surgery, shielding to reduce radiation damage to reproductive organs, and emergency in-vitro fertilisation after controlled ovarian stimulation, with the aim of freezing embryos. The practice of transfering frozen or thawed embryos has been in place for over 25 years, and today is a routine clinical treatment in fertility clinics. Oocytes may also be frozen unfertilised for later thawing and fertilisation by intracytoplasmic sperm injection in vitro. In recent years, oocyte cryopreservation methods have further developed, reaching promising standards. More than 1000 children are born worldwide after fertilisation of frozen and thawed oocytes. Nevertheless, this technique is still considered experimental. In this chapter, we focus on options for fertility preservation still in development that can be offered to women. These include freezing of oocytes and ovarian cortex and the transplantation of ovarian tissue. PMID:22301053

  12. Reproductive Management for Optimal Oocyte Development to Enhance Fertility

    Technology Transfer Automated Retrieval System (TEKTRAN)

    There are multiple steps associated with the ovulatory follicle that affect oocyte growth, fertilization, embryo development and establishment of pregnancy. When estrous cycles are manipulated with assisted reproductive technologies and ovulation induced, some of these variables become more importa...

  13. Functional expression of murine multidrug resistance in Xenopus laevis oocytes

    SciTech Connect

    Castillo, G.; Vera, J.C.; Rosen, O.M. ); Yang, Chiaping Huang; Horwitz, S.B. )

    1990-06-01

    The development of multidrug resistance (MDR) is associated with the overproduction of a plasma membrane glycoprotein, P glycoprotein. Here the authors report the functional expression of a member of the murine MDR family of proteins and show that Xenopus oocytes injected with RNA encoding the mouse mdr1b P glycoprotein develop a MDR-like phenotype. Immunological analysis indicated that oocytes injected with the mdr1b RNA synthesized a protein with the size and immunological characteristics of the mouse mdr1b P glycoprotein. These oocytes exhibited a decreased accumulation of ({sup 3}H)vinblastine and showed an increased capacity to extrude the drug compared to control oocytes not expressing the P glycoprotein. In addition, competition experiments indicated that verapamil, vincristine, daunomycin, and quinidine, but not colchicine, can overcome the rapid drug efflux conferred by the expression of the mouse P glycoprotein.

  14. Physiological and behavioral responses to glucoprivation in the golden hamster.

    PubMed

    Rowland, N

    1983-05-01

    Golden hamsters failed to increase their food intake following food deprivation alone or in combination with insulin or 2-deoxy-D-glucose (2DG) treatment. 2DG also failed to induce feeding in hamsters tested at night. In this latter experiment, there was no effect of 2DG on wheel running or general alertness. Insulin administration significantly decreased plasma levels of glucose and free fatty acids (FFA). 2DG treatment produced a dose-related hyperglycemia associated with increased ketone levels. These data are discussed in terms of cerebral energy status and its relation to food intake and physiological responses.

  15. The Hamster Buccal Pouch Model of Oral Carcinogenesis.

    PubMed

    Nagini, Siddavaram; Kowshik, Jaganathan

    2016-01-01

    The hamster buccal pouch (HBP) carcinogenesis model is one of the most well-characterized animal tumor models used as a prelude to investigate multistage oral carcinogenesis and to assess the efficacy of chemointervention. Hamster buccal pouch carcinomas induced by 7,12-dimethylbenz[a]anthracene (DMBA) show extensive similarities to human oral squamous cell carcinomas. The HBP model offers a number of advantages including a simple and predictable tumor induction procedure, easy accessibility for examination and follow-up of lesions, and reproducibility. This model can be used to test both chemopreventive and chemotherapeutic agents. PMID:27246045

  16. Isolation and identification of normal killer cells from Syrian hamsters

    SciTech Connect

    Matveeva, V.A.; Klyuchareva, T.E.

    1986-09-01

    This paper gives data on isolation of normal killer cells from the blood and various tissues of Syrian hamsters in a Percoll density gradient and their identification on the basis of morphologic criteria and cytotoxic activity (CTA). CTA of the isolated cells was studied in the cytotoxic test with target cells of a human MOLT-4 thymoma cell labeled with /sup 51/Cr. Isolation of large granular lymphocytes from blood, spleen, and bone marrow of Syrian hamsters in Percoll density gradient is shown in the results of five experiments used for cells of each type.

  17. Use of CB hamsters in the study of Treponema pertenue.

    PubMed

    Schell, R F; Le Frock, J L; Babu, J P; Chan, J K

    1979-10-01

    The CB/Ss LAK strain of inbred hamster was used as a model for studies of infection with Treponema pertenue and of acquired resistance to it. When infected, this strain developed cutaneous lesions which lasted for six to seven months, even in the presence of peak titres of antitreponemal antibody. The rate of appearance and resolution of these lesions varied with the size of the inoculum. The infected hamsters' inguinal lymph nodes increased significantly in weight and teemed with treponemes for several weeks. Animals infected for eight or 10 weeks obtained quick resolution of their lesions by treatment with penicillin and were thereafter resistant to reinfection. PMID:509189

  18. [Fertilizing capacity of the ejaculate of nutria (Myocastor coypus) after the removal of the seminal vesicles as evaluated by the penetration test and natural mating].

    PubMed

    Jakubicka, I; Barta, M; Babusík, P

    1989-07-01

    The fertility of male coypu sperm following seminal vesicle extirpation was investigated using the penetration test into the egg of Syrian golden hamster (Mesocricetus auratus). Ejaculates were obtained from five males by means of electro-ejaculation under halothane narcosis. The results of the zona-free hamster eggs (ZFHE) penetration test showed that the ejaculates of all the surgically treated coypu males were fertile and that ZFHE value fluctuated from 54 to 76.6%. The results obtained in experiments with natural mating revealed that the extirpation of male coypu seminal vesicles did not affect their fertility. In total 47 foetuses were found post mortem in ten coypu females covered by surgically treated males, which on average represented 4.7 foetuses per female. PMID:2678717

  19. Intrafollicular transfer of fresh and vitrified immature bovine oocytes.

    PubMed

    Sprícigo, José Felipe W; Sena Netto, Severino Bernardino; Muterlle, Carolle Vieira; Rodrigues, Sarah de Andrade Dias; Leme, Ligiane Oliveira; Guimarães, Ana Luiza; Caixeta, Felippe Manoel Costa; Franco, Maurício Machain; Pivato, Ivo; Dode, Margot Alves Nunes

    2016-11-01

    Embryo production by intrafollicular oocyte transfer (IFOT) represents an alternative for production of a large number of embryos without requiring any hormones and only basic laboratory handling. We aimed to (1) evaluate the efficiency of IFOT using immature oocytes (IFIOT) and (2) compare embryo development after IFIOT using fresh or vitrified immature oocytes. First, six IFIOTs were performed using immature oocytes obtained by ovum pickup. After insemination and uterine flush for embryo recovery, 21.3% of total transferred structures were recovered excluding the recipient's own oocyte or embryo, and of those, 26% (5.5% of transferred cumulus-oocyte complexes [COCs]) were morula or blastocyst. In the second study, we compared fresh and vitrified-warmed immature COCs. Four groups were used: (1) fresh immature COCs (Fresh-Vitro); (2) vitrified immature COCs (Vit-Vitro), with both groups 1 and 2 being matured, fertilized, and cultured in vitro; (3) fresh immature COCs submitted to IFIOT (Fresh-IFIOT); and (4) vitrified immature COCs submitted to IFIOT (Vit-IFIOT). Cumulus-oocyte complexes (n = 25) from Fresh-IFIOT or Vit-IFIOT groups were injected into dominant follicles (>10 mm) of synchronized heifers. After excluding one structure or blastocyst, the recovery rates per transferred oocyte were higher (P < 0.05) for Fresh-IFIOT (47.6%) than for Vit-IFIOT (12.0%). Blastocyst yield per initial oocyte was higher (P < 0.05) for Fresh-Vitro (42.1%) than for Fresh-IFIOT (12.9%). Vit-Vitro presented higher (P < 0.05) embryo development (6.3%), compared to Vit-IFIOT, which did not result in any extra embryo. Although IFOT did not improve developmental competence of vitrified oocytes, we achieved viable blastocysts and pregnancies produced after IFIOT of fresh bovine immature oocytes. Further work on this technique is warranted as an option both for research studies and for clinical bovine embryo production in the absence of laboratory facilities for IVF. PMID

  20. Effects of cryopreservation on the developmental competence, ultrastructure and cytoskeletal structure of porcine oocytes.

    PubMed

    Wu, Caihong; Rui, Rong; Dai, Jianjun; Zhang, Chunyan; Ju, Shiqiang; Xie, Bing; Lu, Xiao; Zheng, Xiaofeng

    2006-11-01

    The purpose of this study was to determine ultrastructural and cytoskeletal changes that result from vitrification of porcine germinal vesicle- (GV-) and meiosis II- (MII-) stage oocytes. To investigate the effects of vitrification on developmental competence, oocytes were divided into three groups: fresh GV-oocytes (control), vitrified GV-oocytes, and vitrified MII-oocytes. In both GV- and MII-oocytes, vitrification resulted in a high proportion with normal morphology (92.4 vs. 94.2%, P > 0.05), while vitrified GV-oocytes yielded a higher survival rate than did vitrified MII-oocytes (56.8 vs. 41.9%, P < 0.05). In vitrified GV-oocytes, 12 of 154 oocytes underwent cleavage after fertilization in vitro, and 6 of these developed to the 8-cell stage, 3 developed to the 16-cell stage, and 3 developed into morulae. No cleavage was obtained from vitrified MII-oocytes. For ultrastructural analysis of oocytes, fresh and vitrified-warmed GV- and MII-oocytes were randomly selected for transmission electron microscopy (TEM). Results showed that vitrification caused various degrees of cryodamage in GV-oocytes. Cumulus cells of some oocytes were separated from the cumulus-oocyte complex (COC), and the zona pellucida adjacent to cumulus cells was fractured. The gap junctions between cumulus cells were ruptured, and many microvilli were disrupted or disappeared. Only homogeneous lipid droplets were observed. After vitrification, cortical granules still lined the oolemma of MII-oocytes. Only morphologically irregular, nonhomogeneous lipid droplets surrounding large vacuoles were found. To examine cytoskeletal structures, fresh and vitrified-warmed MII-oocytes were analyzed by laser-scanning confocal microscopy (LSCM); vitrified-warmed GV-oocytes were cultured for 42-44 hr before LSCM. Of 58 control oocytes, 79.5% displayed normal spindles with chromosomes aligned along the equatorial plate. In vitrified oocytes the percentage with normal spindle organization was decreased

  1. Factors affecting in vitro maturation of alpaca (Lama paco) oocytes.

    PubMed

    Leisinger, Ca; Coffman, Ea; Coutinho da Silva, Ma; Forshey, Bs; Pinto, Crf

    2014-11-10

    The present study utilized a 2×2×2 factorial design examining age (old vs. young), follicle size (≥2mm vs. <2mm) and media supplementation (with or without fetal bovine serum [FBS]) to determine factors that might affect in vitro maturation of alpaca oocytes. We hypothesized that oocytes collected from follicles ≥2mm from young alpacas and incubated in maturation media supplemented with FBS would have greater maturation rates than those incubated in any other factorial combination. Oocytes were collected from the ovaries of 11 young alpacas (<10 years old) and 14 old alpacas (>11 years old). Oocytes were classified as morphologically normal oocytes (MNO) and deemed suitable for incubation if ≥3 compact layers of cumulus cells and a homogeneous, evenly granulated cytoplasm were observed. Oocytes from each group of follicle sizes were incubated separately and halves of each group were randomly divided and incubated 24h in chemically defined maturation media with or without 10% FBS. Maturation was defined as the visualization of a polar body at the end of the incubation period. Overall, a greater proportion of MNO were collected from follicles ≥2mm than that obtained from smaller follicles, 55% (136/247) vs. 29.6% (162/547), respectively (P<0.05). A greater proportion of oocytes reached maturation when collected from ≥2mm follicles 36% (49/136) than from <2mm follicles 8% (13/162) (P<0.05). For oocytes obtained from ≥2mm follicles of old alpacas, a greater proportion reached maturation when incubated in media supplemented with FBS than when incubated without FBS; 57.6% (19/33) vs. 18.2% (6/33), respectively (P<0.05).

  2. Cooperative Transmembrane Penetration of Nanoparticles

    PubMed Central

    Zhang, Haizhen; Ji, Qiuju; Huang, Changjin; Zhang, Sulin; Yuan, Bing; Yang, Kai; Ma, Yu-qiang

    2015-01-01

    Physical penetration of lipid bilayer membranes presents an alternative pathway for cellular delivery of nanoparticles (NPs) besides endocytosis. NPs delivered through this pathway could reach the cytoplasm, thereby opening the possibility of organelle-specific targeting. Herein we perform dissipative particle dynamics simulations to elucidate the transmembrane penetration mechanisms of multiple NPs. Our simulations demonstrate that NPs’ translocation proceeds in a cooperative manner, where the interplay of the quantity and surface chemistry of the NPs regulates the translocation efficiency. For NPs with hydrophilic surfaces, the increase of particle quantity facilitates penetration, while for NPs with partly or totally hydrophobic surfaces, the opposite highly possibly holds. Moreover, a set of interesting cooperative ways, such as aggregation, aggregation-dispersion, and aggregation-dispersion-reaggregation of the NPs, are observed during the penetration process. We find that the penetration behaviors of multiple NPs are mostly dominated by the changes of the NP-membrane force components in the membrane plane direction, in addition to that in the penetration direction, suggesting a different interaction mechanism between the multiple NPs and the membrane compared with the one-NP case. These results provide a fundamental understanding in the underlying mechanisms of cooperative penetration of NPs, and shed light on the NP-based drug and gene delivery. PMID:26013284

  3. Transdermal penetration of UV filters.

    PubMed

    Klinubol, P; Asawanonda, P; Wanichwecharungruang, S P

    2008-01-01

    A penetration study of 2-ethylhexyl-4-methoxycinnamate (EHMC), 4-methyl benzylidenecamphor (MBC), butyl methoxydibenzoylmethane (BMBM), 2-ethylhexyl-2,4,5-trimethoxycinnamate (EHTMC) and di(2-ethylhexyl)-2,4,5-trimethoxybenzalmalonate (TMB) through baby mouse skin (Mus musculus Linn.) was carried out using a vertical Franz diffusion cell. At 4.4 mg/cm(2) coverage of UV filter on the skin, 2.98 +/- 0.38, 1.15 +/- 0.14 and 0.80 +/- 0.28% of the applied EHMC, MBC and BMBM were detected in the receptor fluid at 24 h after application. Penetrations of UV filter in an ethanolic solution and lotion forms were comparable. EHTMC and TMB showed insignificant penetration across the baby mouse skins. Baby mouse skins kept at 4, -20 and -80 degrees C gave similar EHMC penetration results. Penetrations of EHMC, BMBM, EHTMC and TMB across human epidermis were carried out upon 5 volunteers using the suction blister technique. The results also confirmed the significant penetrations of EHMC and BMBM and the insignificant penetrations of EHTMC and TMB.

  4. Projectile penetration into ballistic gelatin.

    PubMed

    Swain, M V; Kieser, D C; Shah, S; Kieser, J A

    2014-01-01

    Ballistic gelatin is frequently used as a model for soft biological tissues that experience projectile impact. In this paper we investigate the response of a number of gelatin materials to the penetration of spherical steel projectiles (7 to 11mm diameter) with a range of lower impacting velocities (<120m/s). The results of sphere penetration depth versus projectile velocity are found to be linear for all systems above a certain threshold velocity required for initiating penetration. The data for a specific material impacted with different diameter spheres were able to be condensed to a single curve when the penetration depth was normalised by the projectile diameter. When the results are compared with a number of predictive relationships available in the literature, it is found that over the range of projectiles and compositions used, the results fit a simple relationship that takes into account the projectile diameter, the threshold velocity for penetration into the gelatin and a value of the shear modulus of the gelatin estimated from the threshold velocity for penetration. The normalised depth is found to fit the elastic Froude number when this is modified to allow for a threshold impact velocity. The normalised penetration data are found to best fit this modified elastic Froude number with a slope of 1/2 instead of 1/3 as suggested by Akers and Belmonte (2006). Possible explanations for this difference are discussed.

  5. Projectile penetration into ballistic gelatin.

    PubMed

    Swain, M V; Kieser, D C; Shah, S; Kieser, J A

    2014-01-01

    Ballistic gelatin is frequently used as a model for soft biological tissues that experience projectile impact. In this paper we investigate the response of a number of gelatin materials to the penetration of spherical steel projectiles (7 to 11mm diameter) with a range of lower impacting velocities (<120m/s). The results of sphere penetration depth versus projectile velocity are found to be linear for all systems above a certain threshold velocity required for initiating penetration. The data for a specific material impacted with different diameter spheres were able to be condensed to a single curve when the penetration depth was normalised by the projectile diameter. When the results are compared with a number of predictive relationships available in the literature, it is found that over the range of projectiles and compositions used, the results fit a simple relationship that takes into account the projectile diameter, the threshold velocity for penetration into the gelatin and a value of the shear modulus of the gelatin estimated from the threshold velocity for penetration. The normalised depth is found to fit the elastic Froude number when this is modified to allow for a threshold impact velocity. The normalised penetration data are found to best fit this modified elastic Froude number with a slope of 1/2 instead of 1/3 as suggested by Akers and Belmonte (2006). Possible explanations for this difference are discussed. PMID:24184862

  6. Non-meiotic chromosome instability in human immature oocytes

    PubMed Central

    Daina, Gemma; Ramos, Laia; Rius, Mariona; Obradors, Albert; del Rey, Javier; Giralt, Magda; Campillo, Mercedes; Velilla, Esther; Pujol, Aïda; Martinez-Pasarell, Olga; Benet, Jordi; Navarro, Joaquima

    2014-01-01

    Aneuploidy has been a major issue in human gametes and is closely related to fertility problems, as it is known to be present in cleavage stage embryos and gestational losses. Pre-meiotic chromosome abnormalities in women have been previously described. The aim of this study is to assess the whole-chromosome complement in immature oocytes to find those abnormalities caused by mitotic instability. For this purpose, a total of 157 oocytes at the germinal vesicle or metaphase I stage, and discarded from IVF cycles, were analysed by CGH. Fifty-six women, between 18 and 45 years old (mean 32.5 years), including 32 IVF patients (25–45 years of age) and 24 IVF oocyte donors (18–33 years of age), were included in the study. A total of 25/157 (15.9%) of the oocytes analysed, obtained from three IVF clinics, contained chromosome abnormalities, including both aneuploidy (24/157) and structural aberrations (9/157). Independently of the maternal age, the incidence of abnormal oocytes which originated before meiosis is 15.9%, and these imbalances were found in 33.9% of the females studied. This work sheds light on the relevance of mitotic instability responsible for the generation of the abnormalities present in human oocytes. PMID:23695274

  7. Improved fertilization rates of human oocytes in coculture.

    PubMed

    Bongso, A; Ng, S C; Fong, C Y; Ratnam, S

    1991-08-01

    Passaged human tubal ampullary epithelial cells from cell lines were used to evaluate fertilization rates and the support of early human embryonic cleavage in vitro. A total of 225 mature oocytes from 32 patients was used in this study. Oocytes from each patient were equally allotted at random to two groups. One hundred thirty-two oocytes were inseminated in the presence of passaged human ampullary cells, while 123 oocytes were inseminated in culture medium alone. Fertilized oocytes were transferred to tubal ampullary cell monolayers and monitored for up to 48 hr. Eighty-five percent of oocytes fertilized in coculture, as compared to 67% in medium alone (P less than 0.01). A higher percentage of good-quality embryos was observed in cocultures than in controls (79 vs 67%; P less than 0.05). Binding of sperm to ampullary monolayer cells was observed. Human ampullary-cell cocultures may be useful to improve fertilization rates and embryonic viability and thus increase take-home baby rates for in vitro fertilization programs.

  8. Frequency of aneuploidy related to age in porcine oocytes.

    PubMed

    Hornak, Miroslav; Jeseta, Michal; Musilova, Petra; Pavlok, Antonin; Kubelka, Michal; Motlik, Jan; Rubes, Jiri; Anger, Martin

    2011-04-27

    It is generally accepted that mammalian oocytes are frequently suffering from chromosome segregation errors during meiosis I, which have severe consequences, including pregnancy loss, developmental disorders and mental retardation. In a search for physiologically more relevant model than rodent oocytes to study this phenomenon, we have employed comparative genomic hybridization (CGH), combined with whole genome amplification (WGA), to study the frequency of aneuploidy in porcine oocytes, including rare cells obtained from aged animals. Using this method, we were able to analyze segregation pattern of each individual chromosome during meiosis I. In contrast to the previous reports where conventional methods, such as chromosome spreads or FISH, were used to estimate frequency of aneuploidy, our results presented here show, that the frequency of this phenomenon was overestimated in porcine oocytes. Surprisingly, despite the results from human and mouse showing an increase in the frequency of aneuploidy with advanced maternal age, our results obtained by the most accurate method currently available for scoring the aneuploidy in oocytes indicated no increase in the frequency of aneuploidy even in oocytes from animals, whose age was close to the life expectancy of the breed.

  9. Frequency of Aneuploidy Related to Age in Porcine Oocytes

    PubMed Central

    Musilova, Petra; Pavlok, Antonin; Kubelka, Michal; Motlik, Jan; Rubes, Jiri; Anger, Martin

    2011-01-01

    It is generally accepted that mammalian oocytes are frequently suffering from chromosome segregation errors during meiosis I, which have severe consequences, including pregnancy loss, developmental disorders and mental retardation. In a search for physiologically more relevant model than rodent oocytes to study this phenomenon, we have employed comparative genomic hybridization (CGH), combined with whole genome amplification (WGA), to study the frequency of aneuploidy in porcine oocytes, including rare cells obtained from aged animals. Using this method, we were able to analyze segregation pattern of each individual chromosome during meiosis I. In contrast to the previous reports where conventional methods, such as chromosome spreads or FISH, were used to estimate frequency of aneuploidy, our results presented here show, that the frequency of this phenomenon was overestimated in porcine oocytes. Surprisingly, despite the results from human and mouse showing an increase in the frequency of aneuploidy with advanced maternal age, our results obtained by the most accurate method currently available for scoring the aneuploidy in oocytes indicated no increase in the frequency of aneuploidy even in oocytes from animals, whose age was close to the life expectancy of the breed. PMID:21556143

  10. Control of Oocyte Growth and Meiotic Maturation in C. elegans

    PubMed Central

    Kim, Seongseop; Spike, Caroline; Greenstein, David

    2013-01-01

    In sexually reproducing animals, oocytes arrest at diplotene or diakinesis and resume meiosis (meiotic maturation) in response to hormones. Chromosome segregation errors in female meiosis I are the leading cause of human birth defects, and age-related changes in the hormonal environment of the ovary are a suggested cause. C. elegans is emerging as a genetic paradigm for studying hormonal control of meiotic maturation. The meiotic maturation processes in C. elegans and mammals share a number of biological and molecular similarities. Major sperm protein (MSP) and luteinizing hormone (LH), though unrelated in sequence, both trigger meiotic resumption using somatic Gαs-adenylate cyclase pathways and soma-germline gap-junctional communication. At a molecular level, the oocyte responses apparently involve the control of conserved protein kinase pathways and post-transcriptional gene regulation in the oocyte. At a cellular level, the responses include cortical cytoskeletal rearrangement, nuclear envelope breakdown, assembly of the acentriolar meiotic spindle, chromosome segregation, and likely changes important for fertilization and the oocyte-to-embryo transition. This chapter focuses on signaling mechanisms required for oocyte growth and meiotic maturation in C. elegans and discusses how these mechanisms coordinate the completion of meiosis and the oocyte-to-embryo transition. PMID:22872481

  11. Discovery and characterization of a new cell-penetrating protein.

    PubMed

    Simeon, Rudo L; Chamoun, Ana Maria; McMillin, Thomas; Chen, Zhilei

    2013-12-20

    We describe a new cell-penetrating protein, B1, capable of delivering conjugated proteins and nucleic acids into mammalian cells. B1 is a 244-amino-acid product of a single-base frameshift in the gene encoding enhanced green fluorescent protein (eGFP). The molecule has a net positive charge of 43 and a very high charge-to-mass ratio of 1.5. eGFP-fused B1 potently penetrates both adherent and suspension cells with >80% of cells taking up the protein when exposed to concentrations as low as 1 μM. The protein was found to cluster in the paranuclear region of TZM-bl cells. Most importantly, we show that B1 not only facilitates cellular uptake but allows biomolecular cargo to reach sites of biological relevance. For example, baby hamster kidney cells underwent DNA recombination when exposed to B1-tagged Cre recombinase at protein concentrations as low as 2.5 μM, indicating potent nuclear delivery of functional protein cargos. Additionally, B1 delivers noncovalently conjugated RNA and DNA across the cell membrane to cytosolic and nuclear sites accessible to the cellular translation and transcription machinery, as gauged by detection of encoded reporter functions, with efficiency comparable to commercially available cationic lipid reagents. B1 appears to utilize cell-surface glycans and multiple competing endocytic pathways to enter and traffic through cells. These studies provide both a new tool for intracellular delivery of biomolecules and insights that could aid in the design of more effective cell penetrating proteins.

  12. Discovery and Characterization of a New Cell-Penetrating Protein

    PubMed Central

    Simeon, Rudo L.; Chamoun, Ana Maria; McMillin, Thomas; Chen, Zhilei

    2013-01-01

    We describe a new cell-penetrating protein, B1, capable of delivering conjugated proteins and nucleic acids into mammalian cells. B1 is a 244-amino-acid product of a single-base frameshift in the gene encoding enhanced green fluorescent protein (eGFP). The molecule has a net positive charge of 43 and a very high charge-to-mass ratio of 1.5. eGFP-fused B1 potently penetrates both adherent and suspension cells with >80% of cells taking up the protein when exposed to concentrations as low as 1 μM. The protein was found to cluster in the paranuclear region of TZM-bl cells. Most importantly, we show that B1 not only facilitates cellular uptake, but allows biomolecular cargo to reach sites of biological relevance. For example, baby hamster kidney cells underwent DNA recombination when exposed to B1-tagged Cre recombinase at protein concentrations as low as 2.5 μM, indicating potent nuclear delivery of functional protein cargos. Additionally, B1 delivers non-covalently conjugated RNA and DNA across the cell membrane to cytosolic and nuclear sites accessible to the cellular translation and transcription machinery, as gauged by detection of encoded reporter functions, with efficiency comparable to commercially available cationic lipid reagents. B1 appears to utilize cell-surface glycans and multiple competing endocytic pathways to enter and traffic through cells. These studies provide both a new tool for intracellular delivery of biomolecules and insights that could aid in the design of more effective cell penetrating proteins. PMID:24047285

  13. 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

  14. Efficient gene targeting in golden Syrian hamsters by the CRISPR/Cas9 system.

    PubMed

    Fan, Zhiqiang; Li, Wei; Lee, Sang R; Meng, Qinggang; Shi, Bi; Bunch, Thomas D; White, Kenneth L; Kong, Il-Keun; Wang, Zhongde

    2014-01-01

    The golden Syrian hamster is the model of choice or the only rodent model for studying many human diseases. However, the lack of gene targeting tools in hamsters severely limits their use in biomedical research. Here, we report the first successful application of the CRISPR/Cas9 system to efficiently conduct gene targeting in hamsters. We designed five synthetic single-guide RNAs (sgRNAs)--three for targeting the coding sequences for different functional domains of the hamster STAT2 protein, one for KCNQ1, and one for PPP1R12C--and demonstrated that the CRISPR/Cas9 system is highly efficient in introducing site-specific mutations in hamster somatic cells. We then developed unique pronuclear (PN) and cytoplasmic injection protocols in hamsters and produced STAT2 knockout (KO) hamsters by injecting the sgRNA/Cas9, either in the form of plasmid or mRNA, targeting exon 4 of hamster STAT2. Among the produced hamsters, 14.3% and 88.9% harbored germline-transmitted STAT2 mutations from plasmid and mRNA injection, respectively. Notably, 10.4% of the animals produced from mRNA injection were biallelically targeted. This is the first success in conducting site-specific gene targeting in hamsters and can serve as the foundation for developing other genetically engineered hamster models for human disease.

  15. Abnormal chromosome behavior in human oocytes which remained unfertilized during human in vitro fertilization.

    PubMed

    Spielmann, H; Krüger, C; Stauber, M; Vogel, R

    1985-09-01

    Chromosomal abnormalities and abnormal embryonic development have previously been observed after human in vitro fertilization (IVF). Chromosomal abnormalities may arise not only after fertilization but even earlier during meiotic maturation of human oocytes in culture. Since chromosomal analysis is simple in oocytes during meiotic maturation, the chromosomal status was analyzed in oocytes which remained unfertilized in a human in vitro fertilization program. In 50 fertilization attempts the chromosomes of 62 unfertilized oocytes could be analyzed; 45 of them were in the process of meiotic maturation. In three oocytes two small polar bodies were observed 16-18 hr after insemination in the absence of fertilization. In one oocyte abnormal chromosome behavior was found during the first meiotic division, and in four oocytes during metaphase of the second meiotic division. These data suggest that chromosomal analysis of unfertilized oocytes in human IVF may improve the understanding human oocyte maturation and fertilization.

  16. Endoplasmic reticulum stress inhibition is a valid therapeutic strategy in vitrifying oocytes.

    PubMed

    Zhao, Nan; Liu, Xue-Jun; Li, Jun-Tao; Zhang, Ling; Fu, Yang; Zhang, Ya-Jie; Chen, Ru-Xin; Wei, Xiao-Qing; Wang, Rui; Wang, Yu; Zhang, Jian-Min

    2015-02-01

    The aim of this study is to determine the link between oocyte cryopreservation and endoplasmic reticulum (ER) stress; whether ER stress inhibition improves the efficiency of oocyte vitrification is also explored. Oocytes from mice were exposure to tauroursodeoxycholic acid (TUDCA, an ER stress inhibitor) or TM (tunicamycin, an ER stress inducer) with or without vitrification. The expressions of X-box binding protein-1 (XBP-1) protein and caspase-12 protein, viability of vitrified-warmed oocytes, and their subsequent embryo competence were measured. The levels of XBP-1 protein and caspase-12 protein expression in vitrified-warmed oocytes were significantly higher than those of fresh control oocytes. TUDCA improved the viability of vitrified-warmed oocytes and their subsequent embryo competence. Mouse oocyte cryopreservation is associated with ER stress, and ER stress inhibition improves the efficiency of oocyte vitrification.

  17. Oocyte development and fecundity type of the Brazilian Snapper Lutjanus alexandrei Moura & Lindeman, 2007 (Perciformes: Lutjanidae).

    PubMed

    Fernandes, C A F; Oliveira, P G V; Oliveira, C H B; Hazin, F H V; Travassos, P

    2016-02-01

    Lutjanid species exhibit multiple spawning behaviour during an extended spawning season in warm months, asynchronous oocyte development and indeterminate fecundity. Although early studies have contributed to knowledge of the reproductive cycle of many species within the group, they have not considered aspects about the number of cortical alveoli oocyte stage throughout maturity phases along spawning season. The latter aspect is also considered very important to confirm indeterminate fecundity hypothesis. In the present study, were analyzed 154 Brazilian snapper Lutjanus alexandrei female gonads obtained from artisanal fisheries in Pernambuco State (Brazil) between October 2010 and March 2011. Were measured oocyte size frequency distribution for maturity phases (developing, spawning capable and actively spawning), and oocyte development stage (unyolked oocytes, cortical alveoli, primary, secondary and tertiary vitellogenic oocytes and hydrated oocytes), and also the oocyte stage frequency during spawning season. The frequency of cortical alveoli oocyte stage was constantly found in the spawning period (>37%), showing slight variation throughout maturity phases. The absence of gap in the oocyte size frequency distribution between primary and secondary oocyte growth stages during spawning season is a strong indicator of continuous oocyte recruitment from reserve stocks. In addition, co-occurrence of tertiary vitellogenic oocytes, hydrated oocytes, post-ovulatory follicles and yellow-brown bodies in the histological sections of ovaries reinforce indeterminate fecundity hypothesis. PMID:26909631

  18. Oocyte development and fecundity type of the Brazilian Snapper Lutjanus alexandrei Moura & Lindeman, 2007 (Perciformes: Lutjanidae).

    PubMed

    Fernandes, C A F; Oliveira, P G V; Oliveira, C H B; Hazin, F H V; Travassos, P

    2016-02-01

    Lutjanid species exhibit multiple spawning behaviour during an extended spawning season in warm months, asynchronous oocyte development and indeterminate fecundity. Although early studies have contributed to knowledge of the reproductive cycle of many species within the group, they have not considered aspects about the number of cortical alveoli oocyte stage throughout maturity phases along spawning season. The latter aspect is also considered very important to confirm indeterminate fecundity hypothesis. In the present study, were analyzed 154 Brazilian snapper Lutjanus alexandrei female gonads obtained from artisanal fisheries in Pernambuco State (Brazil) between October 2010 and March 2011. Were measured oocyte size frequency distribution for maturity phases (developing, spawning capable and actively spawning), and oocyte development stage (unyolked oocytes, cortical alveoli, primary, secondary and tertiary vitellogenic oocytes and hydrated oocytes), and also the oocyte stage frequency during spawning season. The frequency of cortical alveoli oocyte stage was constantly found in the spawning period (>37%), showing slight variation throughout maturity phases. The absence of gap in the oocyte size frequency distribution between primary and secondary oocyte growth stages during spawning season is a strong indicator of continuous oocyte recruitment from reserve stocks. In addition, co-occurrence of tertiary vitellogenic oocytes, hydrated oocytes, post-ovulatory follicles and yellow-brown bodies in the histological sections of ovaries reinforce indeterminate fecundity hypothesis.

  19. Combination of ovarian tissue harvesting and immature oocyte collection for fertility preservation increases preservation yield.

    PubMed

    Hourvitz, A; Yerushalmi, G M; Maman, E; Raanani, H; Elizur, S; Brengauz, M; Orvieto, R; Dor, J; Meirow, D

    2015-10-01

    The aim of this study was to evaluate the safety and efficacy of combined ovarian tissue cryopreservation and oocyte aspiration just before ovarian tissue cryobanking. A retrospective cohort study of fertility preservation patients treated in 2007-2013 in one tertiary centre was performed. A total of 255 cancer patients were admitted for fertility preservation: 142 patients underwent ovarian tissue cryopreservation only (OTC), 56 underwent OTC plus oocyte retrieval from ovarian tissue (OTIVM), nine underwent oocyte aspiration and in-vitro maturation (AIVM) and 48 underwent all three procedures. The total number of oocytes, total number of metaphase II (MII) oocytes, maturation rate, fertilization rate and total number of cryopreserved oocytes between groups were compared. The study found significantly more oocytes (P < 0.001), more MII oocytes (P < 0.001), better maturation rate (P < 0.01) and more cryopreserved oocytes (P < 0.05) with all three compared with OTIVM or OTC. No adverse outcome was observed by performing oocyte retrieval before ovarian resection for cryopreservation. In conclusion, oocyte aspiration just before ovarian tissue cryobanking is safe and gains more oocytes with a better maturation rate than ovarian tissue oocyte cryobanking alone. Better results were obtained with 3 days of stimulation before oocyte retrieval.

  20. Ground Penetrating Radar, Barrow, Alaska

    DOE Data Explorer

    John Peterson

    2015-03-06

    This is 500 MHz Ground Penetrating Radar collected along the AB Line in Intensive Site 1 beginning in October 2012 and collected along L2 in Intensive Site 0 beginning in September 2011. Both continue to the present.

  1. Inspecting the reactor vessel penetrations

    SciTech Connect

    Bodson, F.; Fleming, K.W.

    1995-08-01

    The susceptibility of Alloy 600 to Primary Water Stress Corrosion Cracking (PWSCC) continues to plague nuclear power plants. Recently, the problem of PWSCC cracking has manifested itself in Control Rod Drive Mechanism (CRDM) head penetrations in nuclear plants in Europe. Framatome has been extensively involved in the performance of both inspections and repairs of CRDM head penetrations at Electricite de France (EdF) plants. B and W Nuclear Technologies (BWNT), building on Framatome technology, has developed a fully integrated service package and robotic manipulator to inspect and repair CRDM head penetrations for US utilities. Reactor vessel bottom penetration are also made of Alloy 600 and to tackle this potential PWSCC problem at EdF plants, Framatome has been performing specific inspections in order to detect the appearance of the phenomenon. This paper describes the overall range of inspection techniques and toolings developed to address these issues.

  2. Investigations into Monochloramine Biofilm Penetration

    EPA Science Inventory

    Biofilm in drinking water systems is undesirable. Free chlorine and monochloramine are commonly used as secondary drinking water disinfectants, but monochloramine is perceived to penetrate biofilm better than free chlorine. However, this hypothesis remains unconfirmed by direct b...

  3. The effect of zinc in the form of erythromycin-zinc complex (Zineryt lotion) and zinc acetate on metallothionein expression and distribution in hamster skin.

    PubMed

    Morgan, A J; Lewis, G; Van den Hoven, W E; Akkerboom, P J

    1993-11-01

    The occurrence of zinc-induced synthesis of metallothionein in skin after topical application of the anti-acne drug Zineryt lotion was investigated in hamster ears. The dinitrophenyl hapten-sandwich immunohistochemical method involving a monoclonal anti-metallothionein (MT) antibody (E9) was used to detect and localize zinc-binding MT in the 'treated' and untreated hamster skin. Atomic absorption spectrophotometry and dithizone histochemistry indicated that zinc penetrated the skin more readily, and accumulated more efficiently within the sebaceous glands, when applied to the skin surface as the organo-zinc complex, rather than as the inorganic zinc salt. MT and zinc had similar distributions in hamster skin exposed to the metal. Thus, MT immunoreactivity was especially intense in the sebaceous glands of Zineryt lotion-treated skin, with evidence of nuclear distribution in some cells. Zinc delivered to the sebaceous glands, and released from the organo-complex under the prevailing aqueous conditions, certainly induced MT synthesis; the cysteine-rich protein may protect the pilosebaceous units during the inflammatory phase of acne by scavenging generated oxyradical species.

  4. Cement penetration after patella venting.

    PubMed

    Jones, Christopher W; Lam, Li-On; Butler, Adam; Wood, David J; Walsh, William R

    2009-01-01

    There is a high rate of patellofemoral complications following total knee arthroplasty. Optimization of the cement-bone interface by venting and suction of the tibial plateau has been shown to improve cement penetration. Our study was designed to investigate if venting the patella prior to cementing improved cement penetration. Ten paired cadaver patellae were allocated prior to resurfacing to be vented or non-vented. Bone mineral density (BMD) was measured by DEXA scanning. In vented specimens, a 1.6 mm Kirschner wire was used to breach the anterior cortex at the center. Specimens were resurfaced with standard Profix instrumentation and Versabond bone cement (Smith and Nephew PLC, UK). Cement penetration was assessed from Faxitron and sectioned images by a digital image software package (ImageJ V1.38, NIH, USA). Wilcoxon rank sum test was used to assess the difference in cement penetration between groups. The relationship between BMD and cement penetration was analyzed by Pearson correlation coefficient. There was a strong negative correlation between peak BMD and cement penetration when analyzed independent of experimental grouping (r(2)=-0.812, p=0.004). Wilcoxon rank sum testing demonstrated no significant difference (rank sum statistic W=27, p=0.579) in cement penetration between vented (10.53%+/-4.66; mean+/-std dev) and non-vented patellae (11.51%+/-6.23; mean+/-std dev). Venting the patella using a Kirschner wire does not have a significant effect on the amount of cement penetration achieved in vitro using Profix instrumentation and Versabond cement.

  5. Cement penetration after patella venting.

    PubMed

    Jones, Christopher W; Lam, Li-On; Butler, Adam; Wood, David J; Walsh, William R

    2009-01-01

    There is a high rate of patellofemoral complications following total knee arthroplasty. Optimization of the cement-bone interface by venting and suction of the tibial plateau has been shown to improve cement penetration. Our study was designed to investigate if venting the patella prior to cementing improved cement penetration. Ten paired cadaver patellae were allocated prior to resurfacing to be vented or non-vented. Bone mineral density (BMD) was measured by DEXA scanning. In vented specimens, a 1.6 mm Kirschner wire was used to breach the anterior cortex at the center. Specimens were resurfaced with standard Profix instrumentation and Versabond bone cement (Smith and Nephew PLC, UK). Cement penetration was assessed from Faxitron and sectioned images by a digital image software package (ImageJ V1.38, NIH, USA). Wilcoxon rank sum test was used to assess the difference in cement penetration between groups. The relationship between BMD and cement penetration was analyzed by Pearson correlation coefficient. There was a strong negative correlation between peak BMD and cement penetration when analyzed independent of experimental grouping (r(2)=-0.812, p=0.004). Wilcoxon rank sum testing demonstrated no significant difference (rank sum statistic W=27, p=0.579) in cement penetration between vented (10.53%+/-4.66; mean+/-std dev) and non-vented patellae (11.51%+/-6.23; mean+/-std dev). Venting the patella using a Kirschner wire does not have a significant effect on the amount of cement penetration achieved in vitro using Profix instrumentation and Versabond cement. PMID:19010682

  6. Penetrating nontorso trauma: the extremities

    PubMed Central

    Ball, Chad G.

    2015-01-01

    Summary Similar to penetrating torso trauma, nontorso injuries have undergone a fascinating oscillation between invasive and noninvasive approaches. This article discusses an organized approach to the evaluation and initial treatment of penetrating extremity injuries based on regional anatomy and clinical examination. The approach is reliable, efficient and minimizes both delays in diagnosis and missed injuries. Outpatient follow-up is particularly important for patients with extremity injuries who are discharged home from the emergency department. PMID:26022152

  7. Secondhand smoke induces hepatic apoptosis and fibrosis in hamster fetus.

    PubMed

    Huang, Chien-Wei; Horng, Chi-Ting; Huang, Chih-Yang; Cho, Ta-Hsiung; Tsai, Yi-Chang; Chen, Li-Jeng; Hsu, Tsai-Ching; Tzang, Bor-Show

    2016-09-01

    Secondhand smoke (SHS) is an important health issue worldwide. Inhaling SHS during pregnancy could cause abnormalities in the internal tissues of newborns, which may then impair fetal development and even cause severe intrauterine damage and perinatal death. However, the understanding of cytopathic mechanisms of SHS by maternal passive smoking on fetus liver during pregnancy is still limited. This study analyzed the effects of high-dose SHS (SHSH) on fetus liver using a maternal passive smoking animal model. Experiments showed that hepatic matrix metalloproteinase-9 activity and terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick-end labeling-positive cells were significantly increased in livers from fetuses of hamsters treated with SHSH. Similarly, expressions of both extrinsic and intrinsic apoptotic molecules were significantly higher in livers from fetuses of hamsters exposed to SHSH. Additionally, significantly increased inflammatory proteins, including transforming growth factor β, inducible nitric oxide synthase, and interleukin 1β, and fibrotic signaling molecules, including phosphorylated Smad2/3, SP1, and α-smooth muscle actin, were observed in the fetus livers from hamsters treated with SHSH. This study revealed that SHSH not only increased apoptosis through intrinsic and extrinsic pathways in the livers of fetuses from hamsters exposed to SHSH but also augmented hepatic fibrosis via Smad2/3 signaling.

  8. [Prevention of cholelithiasis with ascorbic acid. Experimental study in hamsters].

    PubMed

    Peraza, M; Méndez, N; Lagarriga, J; Cohen, J; Alcantar, M; Chiprut, R

    1979-01-01

    Cholesterol lithogenesis is the end result of hepatic microsomal enzymatic alterations which determine an increase in cholesterol synthesis (HMG CoA reductase) and a decrease in its transformation into bile salts (7 alpha hydroxylase). Therefore biliary cholesterol excretion is increased while bile salt excretion is diminished. Ascorbic Acid (A.A.) seems capable of reversing those enzymatic derrangements in scorbutic animals. Since hamsters are able to synthesize A.A., we evaluated its effect used in high doses during diet induced lithogenesis. Two groups of 6 weeks old, male hamsters, were fed with a lithogenic diet for 30 days. Group A received the usual amount of A.A. contained in the diet (0.25 mg/day/manster) while group B had supplementary A.A. added to drinking water (5 mg/day/hamster). Thirteen out of twenty of group A (65%) and 5 out of 20 of group B (25%) developed cholesterol calculi (p 0.05). Less stones were found in the gallbladders of hamsters fed with supplementary A.A. It is concluded that A.A. in this model, has an inhibitory effect on lithogenesis. The possible mechanism seems to be related to A.A. influence on the microsomal enzymes involved in lithogenesis. These findings, plus the lack of undesirable secondary effects of supplementary A.A. suggest a potential therapeutic role in human cholelithiasis. PMID:531439

  9. Melatonin production accompanies arousal from daily torpor in Siberian hamsters.

    PubMed

    Larkin, Jennie E; Yellon, Steven M; Zucker, Irving

    2003-01-01

    Arousal from deep hibernation is accompanied by a transient rise of melatonin (Mel) in circulation; there are no comparable analyses of Mel concentrations in species that undergo much shallower, shorter duration episodes of daily torpor. Serum Mel concentrations were determined during arousal from both natural daily torpor and torpor induced by 2-deoxy-D-glucose (2-DG) treatment (2,500 mg/kg, intraperitoneal [IP]); blood samples were drawn from the retro-orbital sinus of anesthetized Siberian hamsters. For animals kept in darkness during torpor, Mel concentrations were highest during early arousal when thermogenesis is maximal, and they decreased as body temperature increased during arousal and returned to baseline once euthermia was reestablished. In hamsters kept in the light during the torpor bout, Mel concentrations were elevated above basal values during arousal, but the response was significantly blunted in comparison with values recorded in darkness. Increased Mel concentrations were detected in hamsters only during arousal from torpor (either natural or 2-DG induced) and were not simply a result of the drug treatment; hamsters that remained euthermic or manifested mild hypothermia after drug treatment maintained basal Mel concentrations. We propose that increased Mel production may reflect enhanced sympathetic activation associated with intense thermogenesis during arousal from torpor rather than an adjustment of the circadian rhythm of Mel secretion.

  10. Neuropeptide Y induces torpor-like hypothermia in Siberian hamsters.

    PubMed

    Paul, Matthew J; Freeman, David A; Park, Jin Ho; Dark, John

    2005-09-01

    Intracerebroventricular (ICV) injections of neuropeptide Y (NPY) are known to decrease body temperature (Tb) of laboratory rats by 1-3 degrees C. Several NPY pathways in the brain terminate in hypothalamic structures involved in energy balance and thermoregulation. Laboratory rats are homeothermic, maintaining Tb within a narrow range. We examined the effect of ICV injected NPY on Tb in the heterothermic Siberian hamster (Phodopus sungorus), a species that naturally undergoes daily torpor in which Tb decreases by as much as 15-20 degrees C. Minimum effective dose was determined in preliminary testing then various doses of NPY were tested in cold-acclimated Siberian hamsters while food was withheld. NPY markedly reduced Tb in the heterothermic Siberian hamster. In addition, the reduction in Tb in 63% of the observations was sufficient to reach the criterion for daily torpor (Tb < 32 degrees C for at least 30 min). Neither the incidence of torpor nor its depth or duration was related to NPY dose. Both likelihood and magnitude of response varied within animals on different test days. NPY decreased 24-h food intake and this was exaggerated in the animals reaching criterion for torpor; the decrease in food intake was positively correlated with the magnitude of the decrease in Tb. The mild hypothermia seen in homeothermic laboratory rats after NPY injected ICV is exaggerated, often greatly, in the heterothermic Siberian hamster. NPY treatment may be activating hypothalamic systems that normally integrate endogenous torpor-producing signals and initiate torpor.

  11. Mutagenicity of instant coffee on cultured Chinese hamster lung cells.

    PubMed

    Nakasato, F; Nakayasu, M; Fujita, Y; Nagao, M; Terada, M; Sugimura, T

    1984-10-01

    Coffee showed mutagenic activity in cultured Chinese hamster lung (CHL) cells as assessed by using diphtheria toxin resistance as a selective marker. Most of the mutagenicity was suppressed in the presence of sodium bisulfite. The contribution of methylglyoxal to the total mutagenicity of coffee was less than 3%.

  12. Hamster bite peritonitis: Pasteurella pneumotropica peritonitis in a dialysis patient.

    PubMed

    Campos, A; Taylor, J H; Campbell, M

    2000-11-01

    We report the first case of Pasteurella pneumotropica peritonitis in a peritoneal dialysis patient. This rare infection was the result of contamination of the dialysis tubing by a pet hamster. We stress the importance of household pets as a source of infection in the peritoneal dialysis population. PMID:11095007

  13. Penetration through the Skin Barrier.

    PubMed

    Nielsen, Jesper Bo; Benfeldt, Eva; Holmgaard, Rikke

    2016-01-01

    The skin is a strong and flexible organ with barrier properties essential for maintaining homeostasis and thereby human life. Characterizing this barrier is the ability to prevent some chemicals from crossing the barrier while allowing others, including medicinal products, to pass at varying rates. During recent decades, the latter has received increased attention as a route for intentionally delivering drugs to patients. This has stimulated research in methods for sampling, measuring and predicting percutaneous penetration. Previous chapters have described how different endogenous, genetic and exogenous factors may affect barrier characteristics. The present chapter introduces the theory for barrier penetration (Fick's law), and describes and discusses different methods for measuring the kinetics of percutaneous penetration of chemicals, including in vitro methods (static and flow-through diffusion cells) as well as in vivo methods (microdialysis and microperfusion). Then follows a discussion with examples of how different characteristics of the skin (age, site and integrity) and of the penetrants (size, solubility, ionization, logPow and vehicles) affect the kinetics of percutaneous penetration. Finally, a short discussion of the advantages and challenges of each method is provided, which will hopefully allow the reader to improve decision making and treatment planning, as well as the evaluation of experimental studies of percutaneous penetration of chemicals.

  14. Penetration through the Skin Barrier.

    PubMed

    Nielsen, Jesper Bo; Benfeldt, Eva; Holmgaard, Rikke

    2016-01-01

    The skin is a strong and flexible organ with barrier properties essential for maintaining homeostasis and thereby human life. Characterizing this barrier is the ability to prevent some chemicals from crossing the barrier while allowing others, including medicinal products, to pass at varying rates. During recent decades, the latter has received increased attention as a route for intentionally delivering drugs to patients. This has stimulated research in methods for sampling, measuring and predicting percutaneous penetration. Previous chapters have described how different endogenous, genetic and exogenous factors may affect barrier characteristics. The present chapter introduces the theory for barrier penetration (Fick's law), and describes and discusses different methods for measuring the kinetics of percutaneous penetration of chemicals, including in vitro methods (static and flow-through diffusion cells) as well as in vivo methods (microdialysis and microperfusion). Then follows a discussion with examples of how different characteristics of the skin (age, site and integrity) and of the penetrants (size, solubility, ionization, logPow and vehicles) affect the kinetics of percutaneous penetration. Finally, a short discussion of the advantages and challenges of each method is provided, which will hopefully allow the reader to improve decision making and treatment planning, as well as the evaluation of experimental studies of percutaneous penetration of chemicals. PMID:26844902

  15. Seasonal aspects of sleep in the Djungarian hamster

    PubMed Central

    Palchykova, Svitlana; Deboer, Tom; Tobler, Irene

    2003-01-01

    Background Changes in photoperiod and ambient temperature trigger seasonal adaptations in the physiology and behaviour of many species, including the Djungarian hamster. Exposure of the hamsters to a short photoperiod and low ambient temperature leads to a reduction of the polyphasic distribution of sleep and waking over the light and dark period. In contrast, a long photoperiod enhances the daily sleep-wake amplitude leading to a decline of slow-wave activity in NREM sleep within the light period. It is unknown whether these changes can be attributed specifically to photoperiod and/or ambient temperature, or whether endogenous components are contributing factors. The influence of endogenous factors was investigated by recording sleep in Djungarian hamsters invariably maintained at a low ambient temperature and fully adapted to a short photoperiod. The second recording was performed when they had returned to summer physiology, despite the maintenance of the 'winter' conditions. Results Clear winter-summer differences were seen in sleep distribution, while total sleep time was unchanged. A significantly higher light-dark cycle modulation in NREM sleep, REM sleep and waking was observed in hamsters in the summer physiological state compared to those in the winter state. Moreover, only in summer, REM sleep episodes were longer and waking bouts were shorter during the light period compared to the dark period. EEG power in the slow-wave range (0.75–4.0 Hz) in both NREM sleep and REM sleep was higher in animals in the summer physiological state than in those in the 'winter' state. In winter SWA in NREM sleep was evenly distributed over the 24 h, while in summer it decreased during the light period and increased during the dark period. Conclusion Endogenous changes in the organism underlie the differences in sleep-wake redistribution we have observed previously in hamsters recorded in a short and long photoperiod. PMID:12756056

  16. Trichloroethylene Metabolism in the Rat Ovary Reduces Oocyte Fertilizability

    PubMed Central

    Wu, Katherine Lily; Berger, Trish

    2007-01-01

    Exposure to trichloroethylene (TCE, an environmental toxicant) reduced oocyte fertilizability in the rat. In vivo, TCE may be metabolized by cytochrome P450 dependent oxidation or glutathione conjugation in the liver or kidneys, respectively. Cytochrome P450 dependent oxidation is the higher affinity pathway. The primary isoform of cytochrome P450 to metabolize TCE in the liver, cytochrome P450 2E1, is present in the rodent ovary. Ovarian metabolism of TCE by the oxidative pathway and the production of reactive oxygen species may occur given the presence of the metabolizing enzyme. The objectives of this study were to define the sensitive interval of oocyte growth to TCE exposure, and to determine if TCE exposure resulted in the formation of ovarian protein carbonyls, an indicator of oxidative damage. Rats were exposed to TCE in drinking water (0.45% TCE (v/v) in 3% Tween) or 3% Tween (vehicle-control) during three 4–5 day intervals of oocyte development preceding ovulation. Oocytes from TCE-exposed females were less fertilizable compared with vehicle-control oocytes. Immunohistochemical labeling of ovaries and Western blotting of ovarian proteins demonstrated TCE treatment induced a greater incidence of protein carbonyls compared with vehicle controls. Protein carbonyl formation in the ovary is consistent with TCE metabolism by the cytochrome P450 pathway. Oxidative damage following ovarian TCE metabolism or the presence of TCE metabolites may contribute to reduced oocyte fertilizability. In summary, these results indicate maturing oocytes are susceptible to very short in vivo exposures to TCE. PMID:17673192

  17. Lipoprotein mediated lipid uptake in oocytes of polychaetes (Annelida).

    PubMed

    Schenk, Sven; Hoeger, Ulrich

    2009-08-01

    The uptake of the 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI)-labeled sex-unspecific Nereis lipoprotein was investigated in oocytes of the nereidid polychaetes Nereis virens and Platynereis dumerilii. The fluorescence label was first observed in endocytic vesicles (<1 microm diameter), which later fused to larger vesicles (2-3 microm); these were finally incorporated into existing unlabeled yolk granules (5-6 microm). In Platynereis oocytes, the fusion of endocytic vesicles was delayed in oocytes at their final stage of development compared with those at an early stage of development. Lipoprotein double-labeled with fluorescein isothiocyanate (FITC) and DiI revealed that both the protein and the lipid moiety remained co-localized during incorporation into the yolk granules of the oocyte. No labeling of the cytoplasmic lipid droplets was observed. In N. virens, unlabeled Nereis lipoprotein was effective as a competitive inhibitor of DiI-labeled Nereis lipoprotein. Ligand blot experiments demonstrated the presence of a lipoprotein receptor with an apparent molecular mass of 120 kDa, which is different from that of the known yolk protein receptor. This indicates the presence, in the polychaete oocyte, of two distinct receptors mediating yolk protein and lipoprotein uptake, respectively. Thus, the sex-unspecific lipoprotein contributes to the lipid supply of the growing oocyte in addition to the known uptake of the yolk-protein-associated lipids. The absence of label in the cytoplasmic lipid droplets, even after prolonged incubation with labeled lipoprotein, suggests that these lipids arise either by the breakdown and resynthesis of lipoprotein-derived lipids and/or by de novo synthesis within the oocyte.

  18. Improvement in in vitro fertilization outcome following in vivo synchronization of oocyte maturation in mice.

    PubMed

    Taiyeb, Ahmed M; Muhsen-Alanssari, Saeeda A; Dees, W L; Ridha-Albarzanchi, Mundhir T; Kraemer, Duane C

    2015-04-01

    Synchronization of oocyte maturation in vitro has been shown to produce higher in vitro fertilization (IVF) rates than those observed in oocytes matured in vitro without synchronization. However, the increased IVF rates never exceeded those observed in oocytes matured in vivo without synchronization. This study was therefore designed to define the effect of in vivo synchronization of oocyte maturation on IVF rates. Mice were superovulated and orally treated with 7.5 mg cilostazol (CLZ), a phosphodiesterase 3A (PDE3A) inhibitor, to induce ovulation of immature oocytes at different stages depending on frequency and time of administration of CLZ. Mice treated with CLZ ovulated germinal vesicle (GV) or metaphase I (MI) oocytes that underwent maturation in vitro or in vivo (i.e. in the oviduct) followed by IVF. Superovulated control mice ovulated mature oocytes that underwent IVF directly upon collection. Ovulated MI oocytes matured in vitro or in vivo had similar maturation rates but significantly higher IVF rates, 2-4 cell embryos, than those observed in control oocytes. Ovulated GV oocytes matured in vitro showed similar maturation rates but significantly higher IVF rates than those observed in control oocytes. However, ovulated GV oocytes matured in vivo had significantly lower IVF rates than those noted in control oocytes. It is concluded that CLZ is able to synchronize oocyte maturation and improve IVF rates in superovulated mice. CLZ may be capable of showing similar effects in humans, especially since temporal arrest of human oocyte maturation with other PDE3A inhibitors in vitro was found to improve oocyte competence level. The capability of a clinically approved PDE3A inhibitor to improve oocyte fertilization rates in mice at doses extrapolated from human therapeutic doses suggests the potential scenario of the inclusion of CLZ in superovulation programs. This may improve IVF outcomes in infertile patients.

  19. Time-Lapse Dynamics of the Mouse Oocyte Chromatin Organisation during Meiotic Resumption

    PubMed Central

    Redi, Carlo Alberto; Zuccotti, Maurizio

    2014-01-01

    In the mammalian oocyte, distinct patterns of centromeres and pericentromeric heterochromatin localisation correlate with the gamete's developmental competence. Mouse antral oocytes display two main types of chromatin organisation: SN oocytes, with a ring of Hoechst-positive chromatin surrounding the nucleolus, and NSN oocytes lacking this ring. When matured to MII and fertilised, only SN oocytes develop beyond the 2-cell, and reach full term. To give detailed information on the dynamics of the SN or NSN chromatin during meiosis resumption, we performed a 9 hr time-lapse observation. The main significant differences recorded are: (1) reduction of the nuclear area only in SN oocytes; (2) ~17 min delay of GVBD in NSN oocytes; (3) chromatin condensation, after GVBD, in SN oocytes; (4) formation of 4-5 CHCs in SN oocytes; (5) increase of the perivitelline space, ~57 min later in NSN oocytes; (6) formation of a rosette-like disposition of CHCs, ~84 min later in SN oocytes; (7) appearance of the MI plate ~40 min later in NSN oocytes. Overall, we described a pathway of transition from the GV to the MII stage that is punctuated of discrete recordable events showing their specificity and occurring with different time kinetics in the two types of oocytes. PMID:24864231

  20. A novel role for DNA methyltransferase 1 in regulating oocyte cytoplasmic maturation in pigs.

    PubMed

    Huan, Yanjun; Xie, Bingteng; Liu, Shichao; Kong, Qingran; Liu, Zhonghua

    2015-01-01

    Maternal factors are required for oocyte maturation and embryo development. To better understand the role of DNA methyltransferase 1 (Dnmt1) in oocyte maturation and embryo development, small interfering RNA (siRNA) was conducted in porcine oocytes. In this study, our results showed that Dnmt1 localized in oocyte cytoplasm and its expression displayed no obvious change during oocyte maturation. When siRNAs targeting Dnmt1 were injected into germinal vesicle (GV) stage oocytes, Dnmt1 transcripts significantly decreased in matured oocytes (P<0.05). After Dnmt1 knockdown in GV stage oocytes, the significant reduction of glutathione content, mitochondrial DNA copy number, glucose-6-phosphate dehydrogenase activity and expression profiles of maternal factors and the severely disrupted distribution of cortical granules were observed in MII stage oocytes (P<0.05), leading to the impaired oocyte cytoplasm. Further study displayed that Dnmt1 knockdown in GV stage oocytes significantly reduced the development of early embryos generated through parthenogenetic activation, in vitro fertilization and somatic cell nuclear transfer (P<0.05). In conclusion, Dnmt1 was indispensable for oocyte cytoplasmic maturation, providing a novel role for Dnmt1 in the regulation of oocyte maturation. PMID:26009894

  1. Cryopreservation of Mammalian Oocytes by Using Sugars: Intra- and extracellular raffinose with small amounts of dimethylsulfoxide yields high cryosurvival, fertilization, and development rates

    PubMed Central

    Eroglu, Ali

    2009-01-01

    Accumulation of intra- and extracellular sugars such as trehalose, glucose, and raffinose is central to survival strategies of a variety of organisms coping with extreme conditions including freezing and almost complete drying. The objective of the present study was to investigate the potential application of intra- and extracellular raffinose in combination with low concentrations of dimethylsulfoxide (Me2SO) to mammalian oocyte cryopreservation. To this end, the fertilization and embryonic development of cryopreserved metaphase II (M II) mouse oocytes were studied in comparison to unfrozen controls. For cryopreservation, M II oocytes were microinjected with 0.1 M raffinose, and then cooled to -196°C in the presence of either 0.3 M raffinose and 0.5 M Me2SO (cryopreservation group 1) or 0.3 M raffinose and 1.0 M Me2SO (cryopreservation group 2). The control groups included untreated oocytes (untreated control) and oocytes microinjected with raffinose, but not frozen (injection control). The post-thaw survival rates were 83.9% and 80.6% for the cryopreservation group 1 and 2, respectively. The fertilization and blastocyst rates in the cryopreservation group 1 (90.0% and 77.8%, respectively) and 2 (94.6% and 72.5%, respectively) were also high and similar to the ones of the injection controls (97.8% and 78.5%, respectively) and untreated controls (98.8% and 83.6%, respectively). These results are consistent with the findings of our earlier studies and support the use of sugars as intra- and extracellular cryoprotectants. Furthermore, the results of the present study indicate that the presence of intra- and extracellular sugars alleviates high concentrations of conventional penetrating cryoprotectants, and thus minimizes their toxicity. PMID:19596315

  2. Cryopreservation of mammalian oocytes by using sugars: Intra- and extracellular raffinose with small amounts of dimethylsulfoxide yields high cryosurvival, fertilization, and development rates.

    PubMed

    Eroglu, Ali

    2010-07-01

    Accumulation of intra- and extracellular sugars such as trehalose, glucose, and raffinose is central to survival strategies of a variety of organisms coping with extreme conditions including freezing and almost complete drying. The objective of the present study was to investigate the potential application of intra- and extracellular raffinose in combination with low concentrations of dimethylsulfoxide (Me(2)SO) to mammalian oocyte cryopreservation. To this end, the fertilization and embryonic development of cryopreserved metaphase II (M II) mouse oocytes were studied in comparison to unfrozen controls. For cryopreservation, M II oocytes were microinjected with 0.1M raffinose, and then cooled to -196 degrees C in the presence of either 0.3M raffinose and 0.5M Me(2)SO (cryopreservation group 1) or 0.3M raffinose and 1.0M Me(2)SO (cryopreservation group 2). The control groups included untreated oocytes (untreated control) and oocytes microinjected with raffinose, but not frozen (injection control). The post-thaw survival rates were 83.9% and 80.6% for the cryopreservation group 1 and 2, respectively. The fertilization and blastocyst rates in the cryopreservation group 1 (90.0% and 77.8%, respectively) and 2 (94.6% and 72.5%, respectively) were also high and similar to the ones of the injection controls (97.8% and 78.5%, respectively) and untreated controls (98.8% and 83.6%, respectively). These results are consistent with the findings of our earlier studies and support the use of sugars as intra- and extracellular cryoprotectants. Furthermore, the results of the present study indicate that the presence of intra- and extracellular sugars alleviates high concentrations of conventional penetrating cryoprotectants, and thus minimizes their toxicity.

  3. Optimized Protocols for In Vitro Maturation of Rat Oocytes Dramatically Improve Their Developmental Competence to a Level Similar to That of Ovulated Oocytes.

    PubMed

    Jiao, Guang-Zhong; Cui, Wei; Yang, Rui; Lin, Juan; Gong, Shuai; Lian, Hua-Yu; Sun, Ming-Ju; Tan, Jing-He

    2016-02-01

    The developmental capacity of in vitro-matured (IVM) oocytes is markedly lower than that of their in vivo-matured (IVO) counterparts, suggesting the need for optimization of IVM protocols in different species. There are few studies on IVM of rat oocytes, and there are even fewer attempts to improve ooplasmic maturation compared to those reported in other species. Furthermore, rat oocytes are well known to undergo spontaneous activation (SA) after leaving the oviduct; however, whether IVM rat oocytes have lower SA rates than IVO oocytes and can potentially be used for nuclear transfer is unknown. In this study, we investigated the effects of maturation protocols on cytoplasmic maturation of IVM rat oocytes and observed the possibility to reduce SA by using IVM rat oocytes. Ooplasmic maturation was assessed using multiple markers, including pre- and postimplantation development, meiotic progression, CG redistribution, redox state, and the expression of developmental potential- and apoptosis-related genes. The results showed that the best protocol consisting of modified Tissue Culture Medium-199 (TCM-199) supplemented with cysteamine/cystine and the cumulus cell monolayer dramatically improved the developmental competence of rat oocytes and supported both pre- and postimplantation development and other ooplasmic maturation makers to levels similar to that observed in ovulated oocytes. Rates of SA were significantly lower in IVM oocytes than in IVO oocytes when observed at the same intervals after nuclear maturation. In conclusion, we have optimized protocols for IVM of rat oocytes that sustain ooplasmic maturation to a level similar to ovulated oocytes. The results suggest that IVM rat oocytes might be used to reduce SA for rat cloning. PMID:26679437

  4. A low-cost automated apparatus for investigating the effects of social defeat in Syrian hamsters.

    PubMed

    Askew, Alicia; González, Fernando

    2014-12-01

    We describe an automated apparatus that can be used to investigate the effects of defeat in hamsters. It consists of a covered alleyway that leads to a box, or arena, where hamsters can be kept separate or allowed to fight. The alleyway is divided into seven equal-sized chambers. Low-power lasers and laser detectors are used to keep track of a hamster's position in the alleyway. A CFL flood lamp placed over the chamber farthest from the arena generates a light gradient in the alleyway that engenders in the subjects a preference for the darker chambers near the arena. A computer automatically records the interruption of the laser beams and yields three measures: average position, the frequency of visits to each chamber, and the frequency of changes in direction of travel in each chamber. The results of a pilot study indicated that when a dominant hamster was placed behind a screened gate in the arena and a subordinate hamster was placed in the alleyway, the subordinate maintained a significantly greater distance from the dominant than did a nondefeated hamster. The subordinate hamster also changed its direction of travel more frequently than did the nondefeated hamster. The results suggest that conditioned fear was elicited in the defeated hamster by proximity to the dominant hamster, an effect that is consistent with published results in which the data were recorded manually or by using commercially available event-tracking software. PMID:24519494

  5. Investigations of oocyte in vitro maturation within a mouse model.

    PubMed

    Chin, Alexis Heng Boon; Chye, Ng Soon

    2004-02-01

    This study attempted to develop a 'less meiotically competent' murine model for oocyte in vitro maturation (IVM), which could more readily be extrapolated to human clinical assisted reproduction. Oocyte meiotic competence was drastically reduced upon shortening the standard duration of in vivo gonadotrophin stimulation from 48 h to 24 h, and by selecting only naked or partially naked germinal vesicle oocytes, instead of fully cumulus enclosed oocyte complexes. With such a less meiotically competent model, only porcine granulosa coculture significantly enhanced the oocyte maturation rate in vitro, whereas no significant enhancement was observed with macaque and murine granulosa coculture. Increased serum concentrations and the supplementation of gonadotrophins, follicular fluid and extracellular matrix gel within the culture medium did not enhance IVM under either cell-free or coculture conditions. Culture medium conditioned by porcine granulosa also enhanced the maturation rate, and this beneficial effect was not diminished upon freeze-thawing. Enhanced IVM in the presence of porcine granulosa coculture did not, however, translate into improved developmental competence, as assessed by in vitro fertilization and embryo culture to the blastocyst stage. PMID:15214575

  6. PTK2b function during fertilization of the mouse oocyte.

    PubMed

    Luo, Jinping; McGinnis, Lynda K; Carlton, Carol; Beggs, Hilary E; Kinsey, William H

    2014-08-01

    Fertilization triggers rapid changes in intracellular free calcium that serve to activate multiple signaling events critical to the initiation of successful development. Among the pathways downstream of the fertilization-induced calcium transient is the calcium-calmodulin dependent protein tyrosine kinase PTK2b or PYK2 kinase. PTK2b plays an important role in fertilization of the zebrafish oocyte and the objective of the present study was to establish whether PTK2b also functions in mammalian fertilization. PTK2b was activated during the first few hours after fertilization of the mouse oocyte during the period when anaphase resumption was underway and prior to the pronuclear stage. Suppression of PTK2b kinase activity in oocytes blocked sperm incorporation and egg activation although sperm-oocyte binding was not affected. Oocytes that failed to incorporate sperm after inhibitor treatment showed no evidence of a calcium transient and no evidence of anaphase resumption suggesting that egg activation did not occur. The results indicate that PTK2b functions during the sperm-egg fusion process or during the physical incorporation of sperm into the egg cytoplasm and is therefore critical for successful development.

  7. Translational regulation of MOS messenger RNA in pig oocytes.

    PubMed

    Dai, Yanfeng; Newman, Barbara; Moor, Robert

    2005-11-01

    The temporal and spatial translation control of stored mRNA in oocytes is regulated by elements in their 3'-untranslated region (3'-UTR). The MOS 3'-UTR in pig oocytes is both heterogeneous (180, 480, or 530 nucleotides), and it contains multiple U-rich elements and extensive A-rich sequences (CA13CA5CA5CA6). We have examined the role of these potential regulatory elements by fusing wild-type or mutant MOS 3'-UTRs to luciferase mRNA and then injecting these chimeric transcripts into oocytes. We draw six main conclusions. First, the length of the MOS 3'-UTR tightly controls the level of translation of luciferase during oocyte maturation. Second, two U-rich (U5A) elements and the hexanucleotide signal (AAUAAA) are required for translation. Third, mutations, duplications, or relocations of the A-rich sequence reduce or block translation. Fourth, the relative importance of the A-rich and U-rich elements in controlling the level of translation differs. Fifth, none of our MOS 3'-UTR manipulations relieved translational repression before germinal vesicle breakdown. Sixth, all the MOS mRNA variants underwent polyadenylation during maturation. Whereas mutations to the hexanucleotide signal block both polyadenylation and translation, mutations to either the A-rich sequence or the U-rich elements block translation without fully blocking polyadenylation. We conclude that MOS mRNA translation in pig oocytes is subject to a more extensive series of controls than that in lower vertebrates.

  8. Differential sensitivity of mouse oocytes to colchicine-induced aneuploidy

    SciTech Connect

    Mailhes, J.B.; Yuan, Z.P.

    1987-01-01

    Unpublished results from our laboratory showed that colchicine increased the incidence of hyperploid mouse metaphase II (MII) oocytes when injected at the same time as human chorionic gonadotrophin (HCG). The objective of the present study was to determine whether the time of administering colchicine influenced the incidence of aneuploidy in MII oocytes. CD-1 mice were given pregnant mare's serum (PMS) and, 48 hr later, HCG. An intraperitoneal injection of 0.2 mg/kg colchicine was given at +4, +2, 0, -2, or -4 hr relative to HCG. Oocytes were collected 17 hr post-HCG and processed, and chromosomes were subsequently C-banded. The percentage of hyperploid oocytes was 0.77, 2.56, 5.71, 7.79, 3.54, and 2.70 for control, +4, +2, 0, -2, or -4 hr pre/post-HCG, respectively. Chi-square analyses of these data demonstrated that colchicine significantly increases the proportion of aneuploid oocytes, and that the relative sensitivity of colchicine-induced aneuploidy depends upon the time that this drug is administered relative to HCG.

  9. Exceptional material requirement for reproduction in mouse oocytes.

    PubMed

    Yu, L; Wang, S F; Zhai, Q Z; Yao, Y Q; Jiang, F; Lu, Y X

    2015-01-01

    Limited information on oocytes and fertilization prevents the efficient therapy of patients with infertility. The most important reason for this lack of understanding is a deficiency in research dedicated to oocytes and fertilization. Currently, we are concerned with the role of nutrition in the process of oocyte development to better understand the relationship between nutrition and infertility. The aim of this study was to explore the relationship between some exceptional materials and infertility to elucidate the role of these materials in oocyte development. We used proteomic analysis to identify numerous nutrition-related proteins in three developmental stages: the germinal vesicle stage, the metaphase II-arrested stage, and the fertilized oocyte-zygote stage. Specific proteins were abundantly expressed during the three stages. These proteins included astacin-like metalloendopeptidase, selenium-binding proteins, and other proteins involved in metabolic and signaling pathways. Other proteins were involved in the citrate cycle, the electron transport chain, the urea cycle, fatty acid metabolism, and the insulin signaling pathway. Almost all these proteins exhibited different expression levels in the three stages. The results of the present study provide a better understanding of the molecular mechanisms of early embryonic development and suggest new treatment methods for infertility. PMID:26600495

  10. The presence of corpus luteum may have a negative impact on in vitro developmental competency of bovine oocytes.

    PubMed

    Hajarian, Hadi; Shahsavari, Mohammad H; Karami-shabankareh, Hamed; Dashtizad, Mojtaba

    2016-03-01

    The aim of the current study was to investigate the effects of the presence or absence of corpus luteum (CL) on in vitro developmental competence of bovine oocytes. In experiment 1, cumulus oocyte complexes (COCs) were collected from slaughterhouse ovaries and divided according to the presence (CL(+) oocytes) or absence (CL(-) oocytes) of a CL in the ovary. Control oocytes (C group) were obtained from ovaries which were not selected toward the presence or absence of CL. All oocytes were submitted to in vitro maturation, fertilization and culture. In experiment 2, the oocytes from the CL(+) and CL(-) ovaries were divided into grown (BCB(+)) and growing (BCB(-)) categories by means of the brilliant cresyl blue (BCB) test. The oocytes from all groups (CL(+)/BCB(+), CL(-)/BCB(+), CL(+)/BCB(-), CL(-)/BCB(-) and control oocytes) were subjected to in vitro embryo production. In experiment 1, the cleavage and blastocyst rates of CL(-) oocytes were higher than those of CL(+) oocytes (83.9% and 43% vs. 69.3% and 22.5%, respectively). In experiment 2, there was less BCB(+) oocytes (more competent oocytes) in the group of CL(+) oocytes than in the group of CL(-) oocytes. Furthermore, developmental competence of all CL(+) oocytes (CL(+)/BCB(+) and CL(+)/BCB(-)) was lower than that of all CL(-) oocytes (CL(-)/BCB(+) and CL(-)/BCB(-)). Thus, the presence of a corpus luteum in the ovary may have negative effects on developmental competence of ipsilateral oocytes. PMID:26952753

  11. Penetration Experiments under Reduced Gravity

    NASA Astrophysics Data System (ADS)

    Krause, C.; Gehlen, M.; Jaquemet, A.; Heller, S.; Sperl, M.; Willnecker, R.

    2013-09-01

    Penetration experiments will find several applications in exploration missions in the near future. Penetrators are common tools for the investigation of physical surface properties. The techniques and theories are widely applied under 1g condition on Earth and the results are used by engineers and scientists. The main contribution to the bearing resistance of a soil is combined of shaft and base resistance [1]. The theories show, that the resistance scales with gravity. Penetration experiments during a parabolic flight campaign have been performed for evaluating this gravity scaling of the bearing resistance in different materials during a parabolic flight campaign in December 2012. The main part of the experiment is composed of a steel rod penetrating into a sample cell. Depth and penetration force are recorded during this process. A sieving mechanism provided the ability of sample preparation during flight. Different compaction regimes of the sample material could be created with a ruttler mounted underneath the sample cell. The parabolic flight campaign consisted of 4 flight days. On each day 13 parabolas with Martian gravity, 12 parabolas with lunar gravity and 6 microgravity parabolas could be performed. Three different sample materials have been examined within the 4 flight days: glass spheres, glass corn and Mojawe sand. The glass spheres and glass corn samples were made of the same material, but with different shape. The Mojawe sand is a natural soil from the Mojawe desert in California (US). The experimental description and the first results will be presented.

  12. Double-Plate Penetration Equations

    NASA Technical Reports Server (NTRS)

    Hayashida, K. B.; Robinson, J. H.

    2000-01-01

    This report compares seven double-plate penetration predictor equations for accuracy and effectiveness of a shield design. Three of the seven are the Johnson Space Center original, modified, and new Cour-Palais equations. The other four are the Nysmith, Lundeberg-Stern-Bristow, Burch, and Wilkinson equations. These equations, except the Wilkinson equation, were derived from test results, with the velocities ranging up to 8 km/sec. Spreadsheet software calculated the projectile diameters for various velocities for the different equations. The results were plotted on projectile diameter versus velocity graphs for the expected orbital debris impact velocities ranging from 2 to 15 km/sec. The new Cour-Palais double-plate penetration equation was compared to the modified Cour-Palais single-plate penetration equation. Then the predictions from each of the seven double-plate penetration equations were compared to each other for a chosen shield design. Finally, these results from the equations were compared with test results performed at the NASA Marshall Space Flight Center. Because the different equations predict a wide range of projectile diameters at any given velocity, it is very difficult to choose the "right" prediction equation for shield configurations other than those exactly used in the equations' development. Although developed for various materials, the penetration equations alone cannot be relied upon to accurately predict the effectiveness of a shield without using hypervelocity impact tests to verify the design.

  13. High-throughput optofluidic system for the laser microsurgery of oocytes

    NASA Astrophysics Data System (ADS)

    Chandsawangbhuwana, Charlie; Shi, Linda Z.; Zhu, Qingyuan; Alliegro, Mark C.; Berns, Michael W.

    2012-01-01

    This study combines microfluidics with optical microablation in a microscopy system that allows for high-throughput manipulation of oocytes, automated media exchange, and long-term oocyte observation. The microfluidic component of the system transports oocytes from an inlet port into multiple flow channels. Within each channel, oocytes are confined against a microfluidic barrier using a steady fluid flow provided by an external computer-controlled syringe pump. This allows for easy media replacement without disturbing the oocyte location. The microfluidic and optical-laser microbeam ablation capabilities of the system were validated using surf clam (Spisula solidissima) oocytes that were immobilized in order to permit ablation of the 5 μm diameter nucleolinus within the oocyte nucleolus. Oocytes were the followed and assayed for polar body ejection.

  14. Growth hormone pathway gene expression varies in porcine cumulus-oocyte complexes during in vitro maturation.

    PubMed

    Zhu, Guiyu; Liu, Shujuan; Jiang, Yunliang; Yang, Honghua; Li, Jinlian

    2008-01-01

    Growth hormone (GH) plays important roles in oocyte development and facilitates the successful production of competent oocytes in many species both in vivo and in vitro. However, the mechanism of GH action on oocyte maturation is not well known. In this paper, the temporospatial messenger ribonucleic acid expression patterns of GH and several other GH-related factors were quantitatively analyzed in porcine cumulus-oocytes complex throughout in vitro maturation (IVM). GH expression was decreased in oocytes during IVM while absent in cumulus cells. GH receptor, insulin-like growth factor-1 (IGF-1), and IGF-1 receptor expressions were also downregulated in oocytes. In cumulus cells, the expression of IGF-1 decreased significantly while IGF-1 receptor expression remained constant. The transcripts of Janus kinase 2 increased in both oocytes and cumulus cells during IVM. The current precise gene expression information provides further evidence to explain the complex network of GH signaling involved in IVM of porcine oocyte.

  15. Ethical Dilemmas for Oocyte Donations: Slippery Slope for Conflicts of Interest.

    PubMed

    Tulay, Pinar

    2016-01-01

    Oocyte donations have increased with improvements in oocyte cryopreservation procedures in recent years. Women with medical conditions that require chemotherapy or radiotherapy have begun to opt for oocyte cryo¬preservation prior to their treatment or to enroll in an oocyte donation program. Alternatively, some women apply for "third-party" oocyte donation programs for nonmedical reasons such as delayed childbearing. Although society seems to accept oocyte donations for medical reasons, it appears that there are still some moral issues surrounding nonmedical oocyte donations. In this review, the ethical aspects of oocyte donations and donors' perspectives are discussed. With developing technologies, the genetic screening of donors has expanded to include diseases. This review explores the ethical issues involved in genetic screening of gamete donors. PMID:27480775

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

    PubMed

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

    2015-07-01

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

  17. Human Oocyte Vitrification: The permeability of metaphase II oocytes to water and ethylene glycol and the appliance toward vitrification

    PubMed Central

    Mullen, Steven F.; Li, Mei; Li, Yuan; Chen, Zi-Jiang; Critser, John K.

    2008-01-01

    Objectives To determine the permeability of human metaphase II oocytes to ethylene glycol and water in the presence of ethylene glycol, and to use this information to develop a method to vitrify human oocytes. Design An incomplete randomized block design was used for this study. Setting A University-affiliated assisted reproductive center. Patients Women undergoing assisted reproduction in the Center for Reproductive Medicine at Shandong University. Interventions Oocytes were exposed to 1.0 molar ethylene glycol in a single step, and photographed during subsequent volume excursions. Main outcome measures A 2-parameter model was employed to estimate the permeability to water and EG. Results Water permeability ranged from 0.15 to 1.17 µm/(min·atm), and ethylene glycol permeability ranged from 1.5 to 30 µm/min between 7 °C at 36 °C. The activation energies for water and ethylene glycol permeability were 14.42 Kcal/mol and 21.20 Kcal/mol, respectively. Conclusions Despite the lower permeability of human MII oocytes to ethylene glycol compared to previously published values for propylene glycol and dimethylsulfoxide, methods to add and remove human oocytes with a vitrifiable concentration of ethylene glycol can be designed which prevent excessive osmotic stress and minimize exposure to high concentrations of this compound. PMID:17681308

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

    PubMed

    Choi, Jung Kyu; 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

  19. Expanding the genetic code in Xenopus laevis oocytes.

    PubMed

    Ye, Shixin; Riou, Morgane; Carvalho, Stéphanie; Paoletti, Pierre

    2013-01-21

    Heterologous expression of ligand-gated ion channels (LGICs) in Xenopus laevis oocytes combined with site-directed mutagenesis has been demonstrated to be a powerful approach to study structure-function relationships. In particular, introducing unnatural amino acids (UAAs) has enabled modifications that are not found in natural proteins. However, the current strategy relies on the technically demanding in vitro synthesis of aminoacylated suppressor tRNA. We report here a general method that circumvents this limitation by utilizing orthogonal aminoacyl-tRNA synthetase (aaRS)/suppressor tRNA(CUA) pairs to genetically encode UAAs in Xenopus oocytes. We show that UAAs inserted in the N-terminal domain of N-methyl-D-aspartate receptors (NMDARs) serve as photo-crosslinkers that lock the receptor in a discrete conformational state in response to UV photo treatment. Our method should be generally applicable to studies of other LGICs in Xenopus oocytes. PMID:23292655

  20. The structure of the mitochondrial cloud of Xenopus laevis oocytes.

    PubMed

    Billett, F S; Adam, E

    1976-12-01

    The ultrastructure of the mitochondrial cloud (Balbiani body) of the pre-vitellogenic oocytes of Xenopus laevis has been examined using transmission and stereoscan electron microscopy. Examination of conventional thin sections confirm previous observations which suggest that the cloud consists essentially of many thousands mitochondria and numerous small vesicles; larger clouds, in oocytes greater than 200 mum in diameter, contain relatively more vesicles. Using a standard electron microscope at 100 kV very long and coursing arrays of mitochondrial profiles can be detected. The presence of very long mitochondrial elements has been confirmed using a high voltage microscope operating at 500-1000 kV. Stereoscan preparations, isolated from pre-vitellogenic oocytes, lend some support to the view that the mitochondrial cloud amy consist of a mass of long filamentous mitochondria and the possibility that there are large continuous regions of mitochondrial material cannot be ruled out.

  1. Translation in the mammalian oocyte in space and time.

    PubMed

    Susor, Andrej; Jansova, Denisa; Anger, Martin; Kubelka, Michal

    2016-01-01

    A hallmark of oocyte development in mammals is the dependence on the translation and utilization of stored RNA and proteins rather than the de novo transcription of genes in order to sustain meiotic progression and early embryo development. In the absence of transcription, the completion of meiosis and early embryo development in mammals relies significantly on maternally synthesized RNAs. Post-transcriptional control of gene expression at the translational level has emerged as an important cellular function in normal development. Therefore, the regulation of gene expression in oocytes is controlled almost exclusively at the level of mRNA and protein stabilization and protein synthesis. This current review is focused on the recently emerged findings on RNA distribution related to the temporal and spatial translational control of the meiotic progression of the mammalian oocyte.

  2. Distributive justice in the allocation of donor oocytes.

    PubMed

    Pennings, G

    2001-02-01

    Due to the shortage of oocyte donors, the waiting lists are lengthening. This raises the ethical question of how the available oocytes should be distributed among candidate recipients. The paper clarifies the ethical structure of the allocation process to find a set of rules that generates decisions that are acceptable for all people involved. The selection includes two steps: admission to the waiting list and ranking of those on the list. The following criteria can be used to decide about the admission of candidates: success rate, health risks, age, parental competence, nationality, primary versus secondary infertility, and capacity to pay. Four criteria may function to rank recipients who should have first priority for receiving oocytes: waiting time, medical urgency, phenotypic matching, and synchronization. The introduction of a point system is defended because it allows balancing of the different ethical principles involved and because it installs an objective system of operating rules which avoid favoritism and personal biases. PMID:11285981

  3. Morphology of preovulatory bovine follicles as related to oocyte maturation.

    PubMed

    de Loos, F A; Bevers, M M; Dieleman, S J; Kruip, T A

    1991-03-01

    Thirty-three preovulatory bovine oocytes and their follicles were collected during the period of final maturation in normally cyclic cows. Cell density of the membrana granulosa, mitotic index of the membrana granulosa, and the occurrence of eosinophilic granulocytes around the basal membrane as well as the maturational stage of the oocyte were determined. Cell density decreased during the period of final maturation. Mitotic indices also decreased after an initial high level in the first hours of the final maturation. Eosinophilic granulocytes were only seen during the last hours of final maturation. The maturational stages of the oocytes were related to distinct maturational stages of the follicular wall as determined by morphological characteristics. We propose a scoring system for the maturity of the follicular wall based on cell density, presence of mitotic figures and the presence of eosinophilic granulocytes outside the vascular compartment. PMID:16726922

  4. Cryopreservation of Mammalian Oocyte for Conservation of Animal Genetics

    PubMed Central

    Prentice, Jennifer R.; Anzar, Muhammad

    2011-01-01

    The preservation of the female portion of livestock genetics has become an international priority; however, in situ conservation strategies are extremely expensive. Therefore, efforts are increasingly focusing on the development of a reliable cryopreservation method for oocytes, in order to establish ova banks. Slow freezing, a common method for cryopreservation of oocytes, causes osmotic shock (solution effect) and intracellular ice crystallization leading to cell damage. Vitrification is an alternative method for cryopreservation in which cells are exposed to a higher concentration of cryoprotectants and frozen with an ultra rapid freezing velocity, resulting in an ice crystal free, solid glass-like structure. Presently, vitrification is a popular method for cryopreservation of embryos. However, vitrification of oocytes is still challenging due to their complex structure and sensitivity to chilling. PMID:20886016

  5. Mars surface penetrator: System description

    NASA Technical Reports Server (NTRS)

    Manning, L. A. (Editor)

    1977-01-01

    A point design of a penetrator system for a Mars mission is described. A strawman payload which is to conduct measurements of geophysical and meteorological parameters is included in the design. The subsystems used in the point design are delineated in terms of power, mass, volume, data, and functional modes. The prospects for survival of the rigors of emplacement are described. Data handling and communications plans are presented to allow consideration of the requirements placed by the penetrator on the orbiter and ground operations. The point design is technically feasible and the payload selection scientifically desirable.

  6. Production of fertile offspring from oocytes grown in vitro by nuclear transfer in cattle.

    PubMed

    Hirao, Yuji; Naruse, Kenji; Kaneda, Masahiro; Somfai, Tamas; Iga, Kosuke; Shimizu, Manabu; Akagi, Satoshi; Cao, Feng; Kono, Tomohiro; Nagai, Takashi; Takenouchi, Naoki

    2013-09-01

    Because of recent advancements in reproductive technology, oocytes have attained an increasingly enriched value as a unique cell population in the production of offspring. The growing oocytes in the ovary are an immediate potential source that serve this need; however, complete oocyte growth before use is crucial. Our research objective was to create in vitro-grown (IVG) oocytes that would have the ability to perform specialized activities, including nuclear reprogramming, as an alternative to in vivo-grown oocytes. Bovine oocyte-granulosa cell complexes with a mean oocyte diameter of approximately 100 μm were cultured on Millicell membrane inserts, with culture medium supplemented with 4% polyvinylpyrrolidone (molecular weight, 360,000), 20 ng/ml androstenedione, 2 mM hypoxanthine, and 5 ng/ml bone morphogenetic protein 7. Oocyte viability after the 14-day culture period was 95%, and there was a 71% increase in oocyte volume. Upon induction of oocyte maturation, 61% of the IVG oocytes extruded a polar body. Eighty-four percent of the reconstructed IVG oocytes that used cumulus cells as donor cells underwent cleavage, and half of them became blastocysts. DNA methylation analyses of the satellite I and II regions of the blastocysts revealed a similar highly methylated status in the cloned embryos derived from in vivo-grown and IVG oocytes. Finally, one of the nine embryos reconstructed from the IVG oocytes developed into a living calf following embryo transfer. Fertility of the offspring was confirmed. In conclusion, the potential of a proportion of the IVG oocytes was comparable to that of in vivo-grown oocytes.

  7. An investigation of the migratory potential of mouse oocytes in vitro.

    PubMed Central

    Mackay, S; Smith, R A; Haig, T

    1992-01-01

    This investigation was undertaken to determine the potential of mouse oocytes for migratory activity using bisected ovaries in vitro. Bisection allowed larger medullary oocytes to be brought nearer to the surface; in this way the migratory potential of all oocytes could be studied. Observations were made following 48 h culture to allow for recovery from any initial traumatic effects resulting from bisection. Ovaries were explanted from fetuses at d 15 postcoitum and from neonatal and postnatal mice (d 1-7, 11, 12 and 14 of life) and examined by scanning and transmission electron microscopy. Oocytes were extruded from the surface and a sequence of events was inferred. Cells superficial to the oocyte sloughed off, exposing the oocytes which showed the migratory phenotype as they emerged onto the surface. Here each oocyte became rounder and was finally extruded, leaving a 'crater'. Scanning electron microscopy of the explant surface allowed counts to be made of emergent oocytes. The number of explants showing emergent oocytes was at a maximum when ovaries were removed at the end of the first week postnatum; the mean number of oocytes emerging from each also peaked at this time. Numbers of migratory oocytes declined in ovaries aged 11 d at explantation and by d 14 only 66% of explants showed oocytes at the surface. The distribution of oocytes of various sizes at the surface suggests that both small cortical oocytes and larger medullary oocytes can express the migratory phenotype. Transmission electron microscopy verified structural integrity of the emerging oocytes and revealed their relationship to underlying cells. Images Fig. 3 Fig. 4 Fig. 5 Fig. 6 Fig. 7 Fig. 8 Fig. 9 Fig. 10 Fig. 11 Fig. 12 Fig. 13 Fig. 14 Fig. 15 Fig. 16 PMID:1304582

  8. Analysis of the oocyte activating capacity and chromosomal complement of round-headed human spermatozoa by their injection into mouse oocytes.

    PubMed

    Rybouchkin, A; Dozortsev, D; Pelinck, M J; De Sutter, P; Dhont, M

    1996-10-01

    Intracytoplasmic sperm injection (ICSI) in the human is a very effective procedure which allows the fertilization of the majority of oocytes even in cases of extreme oligoasthenoteratozoospermia. Round-headed acrosomeless human spermatozoa, however, form an exception to this rule, because in about half of the couples with globozoospermia all oocytes remain unfertilized after injection. The incapacity of the spermatozoon to activate the oocyte following injection of round-headed spermatozoa could be the underlying mechanism. To investigate this hypothesis, activation rates of mouse oocytes injected with spermatozoa from a patient with globozoospermia were compared with those obtained after injection with normal spermatozoa. Of mouse oocytes surviving the injection with donor spermatozoa, 95% underwent activation, compared to none of the 88 mouse oocytes surviving the injection with round-headed spermatozoa. After fixation, prematurely condensed sperm chromosomes were found in these oocytes. Parthenogenetic activation of mouse oocytes (8% ethanol at 40 min after injection) injected with round-headed spermatozoa led to the activation of 96% of oocytes. These oocytes developed normally to the first mitosis and were fixed for analysis of the sperm karyotypes. The incidence of chromosomal abnormalities of round-headed spermatozoa (6%) was similar to that in spermatozoa from a fertile donor (9%). These data provide further information on the basic defect in cases of globozoospermia and demonstrate that globozoospermia is not associated with sperm karyotype abnormalities.

  9. Aflatoxin B1 is toxic to porcine oocyte maturation.

    PubMed

    Liu, Jun; Wang, Qiao-Chu; Han, Jun; Xiong, Bo; Sun, Shao-Chen

    2015-07-01

    As a toxic secondary metabolite of Aspergillus species, Aflatoxin B1 (AFB1) is a major food and feed contaminant in tropical and sub-tropical regions with high temperature and humidity. It has been reported to be toxic to the female reproductive system in laboratory and domestic animals. In the present study, the influence of acute exposure to AFB1 (10 and 50 μM, 44h) on porcine oocyte maturation and its possible mechanism were investigated. The maturation rates of oocytes decreased significantly in the presence of 50 μM of AFB1. Cell cycle analysis showed that most oocytes were arrested at germinal vesicle breakdown or meosis I stage. However, actin assembly, spindle structure and chromosome alignment were not disrupted after exposure to 50 μM AFB1. Further study showed that DNA methylation levels increased in treated oocytes (50 μM). Histone methylation levels were also analysed after treatment (50 μM): H3K27me3 and H3K4me2 levels decreased, whereas H3K9me3 level increased, indicating that epigenetic modification was affected. AFB1 treatment (50 μM) also induced oxidative stress and further led to autophagy, as shown by accumulation of reactive oxygen species, up-regulated LC3 protein expression and increased mRNA levels of ATG3, ATG5 and ATG7. Annexin V-FITC staining assay revealed that AFB1 treatment (50 μM) resulted in oocyte early apoptosis, which was confirmed by increased Bak, Bax, Bcl-xl mRNA levels. Collectively, our results suggest that AFB1 disrupts porcine oocyte maturation through changing epigenetic modifications as well as inducing oxidative stress, excessive autophagy and apoptosis. PMID:25778688

  10. Optimization of Cryoprotectant Loading into Murine and Human Oocytes

    PubMed Central

    Karlsson, Jens O.M.; Szurek, Edyta A.; Higgins, Adam Z.; Lee, Sang R.; Eroglu, Ali

    2014-01-01

    Loading of cryoprotectants into oocytes is an important step of the cryopreservation process, in which the cells are exposed to potentially damaging osmotic stresses and chemical toxicity. Thus, we investigated the use of physics-based mathematical optimization to guide design of cryoprotectant loading methods for mouse and human oocytes. We first examined loading of 1.5 M dimethylsulfoxide (Me2SO) into mouse oocytes at 23°C. Conventional one-step loading resulted in rates of fertilization (34%) and embryonic development (60%) that were significantly lower than those of untreated controls (95% and 94%, respectively). In contrast, the mathematically optimized two-step method yielded much higher rates of fertilization (85%) and development (87%). To examine the causes for oocyte damage, we performed experiments to separate the effects of cell shrinkage and Me2SO exposure time, revealing that neither shrinkage nor Me2SO exposure single-handedly impairs the fertilization and development rates. Thus, damage during one-step Me2SO addition appears to result from interactions between the effects of Me2SO toxicity and osmotic stress. We also investigated Me2SO loading into mouse oocytes at 30°C. At this temperature, fertilization rates were again lower after one-step loading (8%) in comparison to mathematically optimized two-step loading (86%) and untreated controls (96%). Furthermore, our computer algorithm generated an effective strategy for reducing Me2SO exposure time, using hypotonic diluents for cryoprotectant solutions. With this technique, 1.5 M Me2SO was successfully loaded in only 2.5 min, with 92% fertilizability. Based on these promising results, we propose new methods to load cryoprotectants into human oocytes, designed using our mathematical optimization approach. PMID:24246951

  11. Size-specific follicle selection improves mouse oocyte reproductive outcomes.

    PubMed

    Xiao, Shuo; Duncan, Francesca E; Bai, Lu; Nguyen, Catherine T; Shea, Lonnie D; Woodruff, Teresa K

    2015-09-01

    Encapsulated in vitro follicle growth (eIVFG) has great potential to provide an additional fertility preservation option for young women and girls with cancer or other reproductive health threatening diseases. Currently, follicles are cultured for a defined period of time and analyzed as a cohort. However, follicle growth is not synchronous, and culturing follicles for insufficient or excessive times can result in compromised gamete quality. Our objective is to determine whether the selection of follicles based on size, rather than absolute culture time, better predict follicle maturity and oocyte quality. Multilayer secondary mouse follicles were isolated and encapsulated in 0.25% alginate. Follicles were cultured individually either for defined time periods or up to specific follicle diameter ranges, at which point several reproductive endpoints were analyzed. The metaphase II (MII) percentage after oocyte maturation on day 6 was the highest (85%) when follicles were cultured for specific days. However, if follicles were cultured to a terminal diameter of 300-350 μm irrespective of absolute time in culture, 93% of the oocytes reached MII. More than 90% of MII oocytes matured from follicles with diameters of 300-350 μm showed normal spindle morphology and chromosome alignment, 85% of oocytes showed two pronuclei after IVF, 81% developed into the two-cell embryo stage and 38% developed to the blastocyst stage, all significantly higher than the percentages in the other follicle size groups. Our study demonstrates that size-specific follicle selection can be used as a non-invasive marker to identify high-quality oocytes and improve reproductive outcomes during eIVFG. PMID:26116002

  12. Release of ATP induced by hypertonic solutions in Xenopus oocytes

    PubMed Central

    Aleu, Jordi; Martín-Satué, Mireia; Navarro, Piedad; de Lara, Ivanna Pérez; Bahima, Laia; Marsal, Jordi; Solsona, Carles

    2003-01-01

    ATP mediates intercellular communication. Mechanical stress and changes in cell volume induce ATP release from various cell types, both secretory and non-secretory. In the present study, we stressed Xenopus oocytes with a hypertonic solution enriched in mannitol (300 mm). We measured simultaneously ATP release and ionic currents from a single oocyte. A decrease in cell volume, the activation of an inward current and ATP release were coincident. We found two components of ATP release: the first was associated with granule or vesicle exocytosis, because it was inhibited by tetanus neurotoxin, and the second was related to the inward current. A single exponential described the correlation between ATP release and the hypertonic-activated current. Gadolinium ions, which block mechanically activated ionic channels, inhibited the ATP release and the inward current but did not affect the decrease in volume. Oocytes expressing CFTR (cystic fibrosis transmembrane regulator) released ATP under hypertonic shock, but ATP release was significantly inhibited in the first component: that related to granule exocytosis. Since the ATP measured is the balance between ATP release and ATP degradation by ecto-enzymes, we measured the nucleoside triphosphate diphosphohydrolase (NTPDase) activity of the oocyte surface during osmotic stress, as the calcium-dependent hydrolysis of ATP, which was inhibited by more than 50 % in hypertonic conditions. The best-characterized membrane protein showing NTPDase activity is CD39. Oocytes injected with an antisense oligonucleotide complementary to CD39 mRNA released less ATP and showed a lower amplitude in the inward current than those oocytes injected with water. PMID:12562935

  13. Aflatoxin B1 is toxic to porcine oocyte maturation.

    PubMed

    Liu, Jun; Wang, Qiao-Chu; Han, Jun; Xiong, Bo; Sun, Shao-Chen

    2015-07-01

    As a toxic secondary metabolite of Aspergillus species, Aflatoxin B1 (AFB1) is a major food and feed contaminant in tropical and sub-tropical regions with high temperature and humidity. It has been reported to be toxic to the female reproductive system in laboratory and domestic animals. In the present study, the influence of acute exposure to AFB1 (10 and 50 μM, 44h) on porcine oocyte maturation and its possible mechanism were investigated. The maturation rates of oocytes decreased significantly in the presence of 50 μM of AFB1. Cell cycle analysis showed that most oocytes were arrested at germinal vesicle breakdown or meosis I stage. However, actin assembly, spindle structure and chromosome alignment were not disrupted after exposure to 50 μM AFB1. Further study showed that DNA methylation levels increased in treated oocytes (50 μM). Histone methylation levels were also analysed after treatment (50 μM): H3K27me3 and H3K4me2 levels decreased, whereas H3K9me3 level increased, indicating that epigenetic modification was affected. AFB1 treatment (50 μM) also induced oxidative stress and further led to autophagy, as shown by accumulation of reactive oxygen species, up-regulated LC3 protein expression and increased mRNA levels of ATG3, ATG5 and ATG7. Annexin V-FITC staining assay revealed that AFB1 treatment (50 μM) resulted in oocyte early apoptosis, which was confirmed by increased Bak, Bax, Bcl-xl mRNA levels. Collectively, our results suggest that AFB1 disrupts porcine oocyte maturation through changing epigenetic modifications as well as inducing oxidative stress, excessive autophagy and apoptosis.

  14. Clinical definition paper on in vitro maturation of human oocytes.

    PubMed

    Dahan, Michael H; Tan, Seang Lin; Chung, Jintae; Son, Weon-Young

    2016-07-01

    In vitro maturation (IVM) of human oocytes is a reproductive technique which has been practiced for 25 years and is gaining popularity. However, the techniques used for IVM differ substantially across clinics and they result in extremely variable pregnancy rates, partially due to some of these differences in protocols. Such differences include the use in some cycles of hCG triggering prior to oocyte retrieval and the use of a few days of gonadotrophin treatment to support moderate follicle growth. Other important factors are patient selection (including those with polycystic ovaries or decreased ovarian reserve), the number of embryos transferred and cleavage-stage embryo or blastocyst transfer. There are also substantial differences of opinion among clinicians regarding IVM and what it implies. Due to the large variation in protocols, a decision was made to write this paper in an attempt to introduce uniformity when comparing treatments and outcomes of IVM. A clinical definition of IVM was developed: The retrieval of oocytes from small and intermediate sized follicles in an ovary before the largest follicle has surpassed 13 mm in mean diameter. The use of short gonadotrophin stimulation should be acknowledged. However, it should be stated that metaphase II oocytes also have the potential to be collected at that time in the cycles associated with either hCG or GnRH agonist priming. Many feel this is not IVM because some mature oocytes are retrieved, therefore, we recommend renaming this procedure either natural cycle IVF or modified natural cycle IVF (if gonadotrophin stimulation is given) with early triggering, combined with IVM The percentage as well as the absolute number of mature oocytes at retrieval should be indicated. The use of these titles will allow transparency when comparing results of IVM cycles.

  15. Heat and cold acclimation in helium-cold hypothermia in the hamster.

    NASA Technical Reports Server (NTRS)

    Musacchia, X. J.

    1972-01-01

    A study was made of the effects of acclimation of hamsters to high (34-35 C) and low (4-5 C) temperatures for periods up to 6 weeks on the induction of hypothermia in hamsters. Hypothermia was achieved by exposing hamsters to a helox mixture of 80% helium and 20% oxygen at 0 C. Hypothermic induction was most rapid (2-3 hr) in heat-acclimated hamsters and slowest (6-12 hr) in cold-acclimated hamsters. The induction period was intermediate (5-8 hr) in room temperature nonacclimated animals (controls). Survival time in hypothermia was relatable to previous temperature acclimations. The hypothesis that thermogenesis in cold-acclimated hamsters would accentuate resistance to induction of hypothermia was substantiated.

  16. Preparation of Cell Lysate from Mouse Oocytes for Western Blotting Analysis.

    PubMed

    Marangos, Petros

    2016-01-01

    Western Blotting has been used extensively for the identification of the protein factors that regulate mammalian oocyte meiosis. However, the limitations in collecting sufficient numbers of oocytes can hinder the efficiency of the technique. Here we provide a detailed protocol for the accurate preparation of mouse oocyte samples for Western Blotting analysis. PMID:27557583

  17. The signaling pathways by which the Fas/FasL system accelerates oocyte aging

    PubMed Central

    Zhu, Jiang; Lin, Fei-Hu; Zhang, Jie; Lin, Juan; Li, Hong; Li, You-Wei; Tan, Xiu-Wen; Tan, Jing-He

    2016-01-01

    In spite of great efforts, the mechanisms for postovulatory oocyte aging are not fully understood. Although our previous work showed that the FasL/Fas signaling facilitated oocyte aging, the intra-oocyte signaling pathways are unknown. Furthermore, the mechanisms by which oxidative stress facilitates oocyte aging and the causal relationship between Ca2+ rises and caspase-3 activation and between the cell cycle and apoptosis during oocyte aging need detailed investigations. Our aim was to address these issues by studying the intra-oocyte signaling pathways for Fas/FasL to accelerate oocyte aging. The results indicated that sFasL released by cumulus cells activated Fas on the oocyte by increasing reactive oxygen species via activating NADPH oxidase. The activated Fas triggered Ca2+ release from the endoplasmic reticulum by activating phospholipase C-γ pathway and cytochrome c pathway. The cytoplasmic Ca2+ rises activated calcium/calmodulin-dependent protein kinase II (CaMKII) and caspase-3. While activated CaMKII increased oocyte susceptibility to activation by inactivating maturation-promoting factor (MPF) through cyclin B degradation, the activated caspase-3 facilitated further Ca2+ releasing that activates more caspase-3 leading to oocyte fragmentation. Furthermore, caspase-3 activation and fragmentation were prevented in oocytes with a high MPF activity, suggesting that an oocyte must be in interphase to undergo apoptosis. PMID:26869336

  18. The growth of the oocyte and follicle in the ovaries of monotremes and marsupials.

    PubMed

    Lintern-Moore, S; Moore, G P; Tyndale-Biscoe, C H; Poole, W E

    1976-07-01

    Oocyte growth in relation to follicular growth in marsupials conforms to the uniform biphasic pattern characteristic of eutherian mammals. However, the oocyte of the marsupial is larger and completes growth in a correspondingly larger follicle. At the completion of oocyte growth the follicle, like that of eutherians, shows no appreciable accumulation of follicular fluid. The ratio of oocyte/follicular diameter in marsupials at this stage is 0.59, a value close to that of 0.55 for eutherian mammals. The oocyte nucleus of the marsupial is also significantly larger than that of the eutherian, but the oocyte nucleolus is approximately the same diameter. By contrast, the growth of the oocyte in relation to the follicle in monotremes in linear throughout the whole period of follicular growth. However, like eutherians and marsupials oocyte nuclear and oocyte nucleolar growth are arrested in the early stages of follicular growth. The diameters of the oocyte nucleus and nucleolus at the completion of growth are approximately twice those found in marsupials. It is concluded that the growth patterns of the oocyte nucleus and nucleolus are common to all mammals but that the biphasic pattern of oocyte and follicular growth which is characteristic of eutherian and marsupial ovaries is not found in the monotremes.

  19. Regulation of the G2/M Transition in Rodent Oocytes

    PubMed Central

    Downs, Stephen M.

    2014-01-01

    Regulation of maturation in meiotically competent mammalian oocytes is a complex process involving the carefully coordinated exchange of signals between the somatic and germ cell compartments of the ovarian follicle via paracrine and cell-cell coupling pathways. This review highlights recent advances in our understanding of how such signaling controls both meiotic arrest and gonadotropin-triggered meiotic resumption in competent oocytes and relates them to the historical context. Emphasis will be on rodent systems, where many of these new findings have taken place. A regulatory scheme is then proposed that integrates this information into an overall framework for meiotic regulation that demonstrates the complex interplay between different follicular compartments. PMID:20578061

  20. Artificial intelligence techniques for embryo and oocyte classification.

    PubMed

    Manna, Claudio; Nanni, Loris; Lumini, Alessandra; Pappalardo, Sebastiana

    2013-01-01

    One of the most relevant aspects in assisted reproduction technology is the possibility of characterizing and identifying the most viable oocytes or embryos. In most cases, embryologists select them by visual examination and their evaluation is totally subjective. Recently, due to the rapid growth in the capacity to extract texture descriptors from a given image, a growing interest has been shown in the use of artificial intelligence methods for embryo or oocyte scoring/selection in IVF programmes. This work concentrates the efforts on the possible prediction of the quality of embryos and oocytes in order to improve the performance of assisted reproduction technology, starting from their images. The artificial intelligence system proposed in this work is based on a set of Levenberg-Marquardt neural networks trained using textural descriptors (the local binary patterns). The proposed system was tested on two data sets of 269 oocytes and 269 corresponding embryos from 104 women and compared with other machine learning methods already proposed in the past for similar classification problems. Although the results are only preliminary, they show an interesting classification performance. This technique may be of particular interest in those countries where legislation restricts embryo selection. One of the most relevant aspects in assisted reproduction technology is the possibility of characterizing and identifying the most viable oocytes or embryos. In most cases, embryologists select them by visual examination and their evaluation is totally subjective. Recently, due to the rapid growth in our capacity to extract texture descriptors from a given image, a growing interest has been shown in the use of artificial intelligence methods for embryo or oocyte scoring/selection in IVF programmes. In this work, we concentrate our efforts on the possible prediction of the quality of embryos and oocytes in order to improve the performance of assisted reproduction technology

  1. Estrogen receptor immunoreactivity in prepubertal and adult male Syrian hamsters.

    PubMed

    Romeo, R D; Diedrich, S L; Sisk, C L

    1999-04-23

    Estrogen and estrogen receptors (ER) are involved in the expression of steroid-dependent male sexual behavior and negative feedback regulation of the hypothalamic-pituitary-gonadal axis. The purpose of the present experiment was to determine whether there are pubertal changes in ER expression in brain that are correlated with pubertal changes in responsiveness to steroid negative feedback and behavioral activation. We found equivalent numbers of ER-immunoreactive (ER-ir) cells in castrated prepubertal and adult male hamsters in nuclei that comprise the neural circuit that mediate male sexual behavior. Therefore, increases in the number of cells in these nuclei that express ER are not correlated with the increased behavioral responsiveness to steroid hormone shown by hamsters after puberty. The number of ER-ir cells in the ventral medial hypothalamus was less in adults compared with juveniles. This pubertal decrease in ER expression is correlated with the decreased responsiveness to steroid negative feedback in the adult.

  2. Sarcolemmal phospholipid N-methylation in genetically determined hamster cardiomyopathy

    SciTech Connect

    Okumura, K.; Panagia, V.; Jasmin, G.; Dhalla, N.S.

    1987-02-27

    The heart sarcolemmal phosphatidylethanolamine N-methylation in UM-X7.1 strain of cardiomyopathic hamsters was examined by using 0.055, 10 and 150 microM S-adenosyl-L-(methyl-/sup 3/H) methionine as methyl donor for sites I, II and III, respectively. In comparison with control values, methylation activities at site I was increased in 40, 120 and 250 days old cardiomyopathic hamsters. On the other hand, methylation activities at sites II and III in 120 and 250 days old cardiomyopathic animals were depressed without any change in the 40 days old group. The alterations in N-methylation activities were associated with kinetic changes in apparent Vmax values without any changes in the apparent Km. These results indicate a defect in the phospholipid N-methylation process in heart sarcolemma during the development of genetically determined cardiomyopathy.

  3. Cystolithiasis in a Syrian hamster: a different outcome.

    PubMed

    Petrini, D; Di Giuseppe, M; Deli, G; De Caro Carella, C

    2016-01-01

    A 14-month-old intact male Syrian hamster was admitted for lethargy and hematuria. A total body radiographic image and abdominal ultrasonography showed the presence of a vesical calculus. During cystotomy, a sterile urine sample was obtained and sent to the diagnostic laboratory along with the urolith for analysis. Urine culture was found negative for bacterial growth, and the urolith was identified as a calcium-oxalate stone. Diet supplementation with palmitoylethanolamide, glucosamine and hesperidin was adopted the day after discharge. One year follow up revealed no presence of vesical calculi. Although this is the report of a single clinical case, this outcome differs from the results reported in the literature characterized by recurrences after few months. Considering the positive outcome and the beneficial properties of palmitoylethanolamide, glucosamine, and hesperidin, these nutritional elements in Syrian hamsters, are recommended to reduce recurrence after surgical treatment of urolithiasis. PMID:27540515

  4. Cystolithiasis in a Syrian hamster: a different outcome

    PubMed Central

    Petrini, D.; Di Giuseppe, M.; Deli, G.; De Caro Carella, C.

    2016-01-01

    A 14-month-old intact male Syrian hamster was admitted for lethargy and hematuria. A total body radiographic image and abdominal ultrasonography showed the presence of a vesical calculus. During cystotomy, a sterile urine sample was obtained and sent to the diagnostic laboratory along with the urolith for analysis. Urine culture was found negative for bacterial growth, and the urolith was identified as a calcium-oxalate stone. Diet supplementation with palmitoylethanolamide, glucosamine and hesperidin was adopted the day after discharge. One year follow up revealed no presence of vesical calculi. Although this is the report of a single clinical case, this outcome differs from the results reported in the literature characterized by recurrences after few months. Considering the positive outcome and the beneficial properties of palmitoylethanolamide, glucosamine, and hesperidin, these nutritional elements in Syrian hamsters, are recommended to reduce recurrence after surgical treatment of urolithiasis. PMID:27540515

  5. Cleft palate development in hamster embryos following triamcinolone treatment.

    PubMed Central

    Shah, R M

    1979-01-01

    Development of the palate was studied in normal and triamcionolone-treated hamster fetuses. The results demonstrated that normal palatogenesis was completed between days 12 and 13 of gestation. Following triamcinolone treatment the reorientation of the palatal shelves was delayed before there was any general retardation of fetal growth (as indicated by crown-rump length and body weight). Since triamcinolone affected palatogenesis at an earlier stage than hydrocortisone, the view that the former is a more potent teratogen was supported. Chronological age, fetal weight and crown-rump length were reliable predictors of normal palatogenesis in the hamster, whereas the numerical morphological rating systems were not. Neither measures of general fetal growth, nor numerical rating, were useful in predicting the stages of experimentally induced cleft palate, since triamcinolone appears to be site-specific, and the drug does not produce a general retardation of embryonic development. PMID:575531

  6. Hibernation, stress, intestinal functions, and catecholoamine turnover rate in hamsters and gerbils

    NASA Technical Reports Server (NTRS)

    Musacchia, X. J.

    1973-01-01

    Bioenergetic studies on hamsters during depressed metabolic states are reported. External support of blood glucose extended the survival times of hibernating animals. Radioresistance increased in hibernating as well as in hypothermic hamsters. Marked changes in hamster catecholamine turnover rates were observed during acclimatization to high temperature stress. High radioresistance levels of the gerbil gastrointestinal system were attributed in part to the ability of the gut to maintain functional integrity.

  7. Metabolic influences on circadian rhythmicity in Siberian and Syrian hamsters exposed to long photoperiods.

    PubMed

    Challet, E; Kolker, D E; Turek, F W

    2000-01-01

    Calorie restriction and other situations of reduced glucose availability in rodents alter the entraining effects of light on the circadian pacemaker located in the suprachiasmatic nuclei. Siberian and Syrian hamsters are photoperiodic species that are sexually active when exposed to long summer-like photoperiods, while both species show opposite changes in body mass when transferred from long to short or short to long days. Because metabolic cues may fine tune the photoperiodic responses via the suprachiasmatic nuclei, we tested whether timed calorie restriction can alter the photic synchronization of the light-entrainable pacemaker in these two hamster species exposed to long photoperiods. Siberian and Syrian hamsters were exposed to 16 h:8 h light:dark cycles and received daily hypocaloric (75% of daily food intake) or normocaloric diet (100% of daily food intake) 4 h after light onset. Four weeks later, hamsters were transferred to constant darkness and fed ad libitum. The onset of the nocturnal pattern of locomotor activity was phase advanced by 1.5 h in calorie-restricted Siberian hamsters, but not in Syrian hamsters. The lack of phase change in calorie-restricted Syrian hamsters was also observed in individuals exposed to 14 h:10 h dim light:dark cycles and fed with lower hypocaloric food (i.e. 60% of daily food intake) 2 h after light onset. Moreover, in hamsters housed in constant darkness and fed ad lib., light-induced phase shifts of the locomotor activity in Siberian hamsters, but not in Syrian hamsters were significantly reduced when glucose utilization was blocked by pretreatment with 500 mg/kg i.p. 2-deoxy-D-glucose. Taken together, these results show that the photic synchronization of the light-entrainable pacemaker can be modulated by metabolic cues in Siberian hamsters, but not in Syrian hamsters maintained on long days.

  8. Molecular and Immunological Characterization of the First Allergenic Lipocalin in Hamster

    PubMed Central

    Torres, José Alberto; de las Heras, Manuel; Maroto, Aroa Sanz; Vivanco, Fernando; Sastre, Joaquín; Pastor-Vargas, Carlos

    2014-01-01

    The most frequent pet allergy is to cat and dog, but in recent years, it has become increasingly popular to have other pets, and the risk of exposure to new allergens is more prevalent. The list of new pets includes hamsters, and one of the most popular hamsters is the Siberian hamster (Phodopus sungorus). The aim of this study was the characterization and cloning of the major allergen from this hamster. The study of its allergenicity and cross-reactivity could improve the specific diagnosis and treatment for hamster-allergic patients. Thirteen Siberian hamster-allergic patients were recruited at the outpatient clinic. Protein extracts were prepared from the hair, urine, and salivary glands of four hamster species (European, golden, Siberian, and Roborovski). IgE-binding proteins were detected by immunoblotting and identified by mass spectrometry. The recombinant protein was produced in Escherichia coli and then purified by metal chelate affinity chromatography. The allergenic properties of the recombinant protein were tested by ELISA and immunoblotting, and biological activity was tested according to capacity for basophil activation. Three IgE-binding proteins were identified in extracts obtained from Siberian hamster hair, urine, and salivary glands. All proteins corresponded to the same protein, which was identified as a lipocalin. This lipocalin had no cross-reactivity with common and golden hamsters. The recombinant allergen was cloned and purified, showing similar IgE reactivity in vitro to Siberian hamster protein extracts. Also, the recombinant allergen was capable of producing biological activation in vivo. The major Siberian hamster allergen was cloned, and allergenic properties were characterized, providing a new tool for specific diagnosis of allergy to Siberian hamster. PMID:24993820

  9. Meiotic maturation and developmental capability of ovine oocytes at germinal vesicle stage following vitrification using different cryodevices.

    PubMed

    Quan, Guo Bo; Wu, Guo Quan; Wang, Ya Jing; Ma, Yuan; Lv, Chun Rong; Hong, Qiong Hua

    2016-02-01

    In order to assess effects of vitrification on ovine oocytes at the germinal vesicle (GV) stage, the conventional plastic straw (CS), the open-pulled straw (OPS), and Cryoloop were used to vitrify ovine oocytes. Oocytes were randomly divided into five groups: (1) Control; (2) Oocytes exposed to vitrification and dilution solutions without any cryopreservation (toxicity); (3) Oocytes vitrified using CS (CS); (4) Oocytes vitrified using OPS (OPS), and (5) Oocytes vitrified using Cryoloop (Cryoloop). The viability, cumulus cell expansion, nuclear maturation after in vitro maturation (IVM), and developmental capability of vitrified oocytes following parthenogenetic activation (PA) or in vitro fertilization (IVF) were assessed. The pretreatment in the vitrification and dilution solutions without any freezing or thawing did not adversely influence oocytes. The viability of vitrified oocytes were significantly declined compared to unfrozen oocytes (P < 0.05). The viability of oocytes vitrified using open-pulled straws or Cryoloop was significantly higher than that in the CS group (P < 0.05). After IVM, the percentage of oocytes reaching to the metaphase II (MII) stage was significantly higher with Cryoloop and OPS following by CS. However, the in vitro maturing percentage of vitrified oocytes was significantly less than that of unfrozen oocytes (P < 0.05). After PA, the developmental capability of vitrified oocytes was significantly decreased compared to unfrozen oocytes. The cleavage rate of oocytes vitrified using conventional plastic straws was significantly less than those of the other freezing groups (P < 0.05). The cleaving capability of oocytes vitrified using Cryoloop was significantly increased compared to the OPS group. However, there was no significant difference existing amongst the freezing groups as concerning the blastocyst rate. Following IVF, the developmental capability of vitrified oocytes was severely damaged compared to that of unfrozen

  10. A Golden Hamster Model for Human Acute Nipah Virus Infection

    PubMed Central

    Wong, K. Thong; Grosjean, Isabelle; Brisson, Christine; Blanquier, Barissa; Fevre-Montange, Michelle; Bernard, Arlette; Loth, Philippe; Georges-Courbot, Marie-Claude; Chevallier, Michelle; Akaoka, Hideo; Marianneau, Philippe; Lam, Sai Kit; Wild, T. Fabian; Deubel, Vincent

    2003-01-01

    A predominantly pig-to-human zoonotic infection caused by the novel Nipah virus emerged recently to cause severe morbidity and mortality in both animals and man. Human autopsy studies showed the pathogenesis to be related to systemic vasculitis that led to widespread thrombotic occlusion and microinfarction in most major organs especially in the central nervous system. There was also evidence of extravascular parenchymal infection, particularly near damaged vessels (Wong KT, Shieh WJ, Kumar S, Norain K, Abdullah W, Guarner J, Goldsmith CS, Chua KB, Lam SK, Tan CT, Goh KJ, Chong HT, Jusoh R, Rollin PE, Ksiazek TG, Zaki SR, Nipah Virus Pathology Working Group: Nipah virus infection: Pathology and pathogenesis of an emerging paramyxoviral zoonosis. Am J Pathol 2002, 161:2153–2167). We describe here a golden hamster (Mesocricetus auratus) model that appears to reproduce the pathology and pathogenesis of acute human Nipah infection. Hamsters infected by intranasal or intraperitoneal routes died within 9 to 29 days or 5 to 9 days, respectively. Pathological lesions were most severe and extensive in the hamster brain. Vasculitis, thrombosis, and more rarely, multinucleated endothelial syncytia, were found in blood vessels of multiple organs. Viral antigen and RNA were localized in both vascular and extravascular tissues including neurons, lung, kidney, and spleen, as demonstrated by immunohistochemistry and in situ hybridization, respectively. Paramyxoviral-type nucleocapsids were identified in neurons and in vessel walls. At the terminal stage of infection, virus and/or viral RNA could be recovered from most solid organs and urine, but not from serum. The golden hamster is proposed as a suitable model for further studies including pathogenesis studies, anti-viral drug testing, and vaccine development against acute Nipah infection. PMID:14578210

  11. Photoperiodic Influences on Ultradian Rhythms of Male Siberian Hamsters

    PubMed Central

    Prendergast, Brian J.; Zucker, Irving

    2012-01-01

    Seasonal changes in mammalian physiology and behavior are proximately controlled by the annual variation in day length. Long summer and short winter day lengths markedly alter the amplitude of endogenous circadian rhythms and may affect ultradian oscillations, but the threshold photoperiods for inducing these changes are not known. We assessed the effects of short and intermediate day lengths and changes in reproductive physiology on circadian and ultradian rhythms of locomotor activity in Siberian hamsters. Males were maintained in a long photoperiod from birth (15 h light/day; 15 L) and transferred in adulthood to 1 of 7 experimental photoperiods ranging from 14 L to 9 L. Decreases in circadian rhythm (CR) robustness, mesor and amplitude were evident in photoperiods ≤14 L, as were delays in the timing of CR acrophase and expansion of nocturnal activity duration. Nocturnal ultradian rhythms (URs) were comparably prevalent in all day lengths, but 15 L markedly inhibited the expression of light-phase URs. The period (τ’), amplitude and complexity of URs increased in day lengths ≤13 L. Among hamsters that failed to undergo gonadal regression in short day lengths (nonresponders), τ’ of the dark-phase UR was longer than in photoresponsive hamsters; in 13 L the incidence and amplitude of light-phase URs were greater in hamsters that did not undergo testicular regression. Day lengths as long as 14 L were sufficient to trigger changes in the waveform of CRs without affecting UR waveform. The transition from a long- to a short-day ultradian phenotype occurred for most UR components at day lengths of 12 L–13 L, thereby establishing different thresholds for CR and UR responses to day length. At the UR-threshold photoperiod of 13 L, differences in gonadal status were largely without effect on most UR parameters. PMID:22848579

  12. Circadian arrhythmia dysregulates emotional behaviors in aged Siberian hamsters

    PubMed Central

    Prendergast, Brian J.; Onishi, Kenneth G.; Patel, Priyesh N.; Stevenson, Tyler J.

    2014-01-01

    Emotional behaviors are influenced by the circadian timing system. Circadian disruptions are associated with depressive-like symptoms in clinical and preclinical populations. Circadian rhythm robustness declines markedly with aging and may contribute to susceptibility to emotional dysregulation in aged individuals. The present experiments used a model of chronic circadian arrhythmia generated noninvasively, via a series of circadian-disruptive light treatments, to investigate interactions between circadian desynchrony and aging on depressive- and anxiety-like behaviors, and on limbic neuroinflammatory gene expression that has been linked with emotionality. We also examined whether a social manipulation (group housing) would attenuate effects of arrhythmia on emotionality. In aged (14-18 months of age) male Siberian hamsters, circadian arrhythmia increased behavioral despair and decreased social motivation, but decreased exploratory anxiety. These effects were not evident in younger (5-9 months of age) hamsters. Social housing (3-5 hamsters/cage) abolished the effects of circadian arrhythmia on emotionality. Circadian arrhythmia alone was without effect on hippocampal or cortical interleukin-1β (IL-1β) and indoleamine 2,3-dioxygenase (Ido) mRNA expression in aged hamsters, but social housing decreased hippocampal IL-1β and Ido mRNAs. The data demonstrate that circadian disruption can negatively impact affective state, and that this effect is pronounced in older individuals. Although clear associations between circadian arrhythmia and constitutive limbic proinflammatory activity were not evident, the present data suggest that social housing markedly inhibits constitutive hippocampal IL-1β and Ido activity, which may contribute to the ameliorating effects of social housing on a number of emotional behaviors. PMID:24333374

  13. Circadian arrhythmia dysregulates emotional behaviors in aged Siberian hamsters.

    PubMed

    Prendergast, Brian J; Onishi, Kenneth G; Patel, Priyesh N; Stevenson, Tyler J

    2014-03-15

    Emotional behaviors are influenced by the circadian timing system. Circadian disruptions are associated with depressive-like symptoms in clinical and preclinical populations. Circadian rhythm robustness declines markedly with aging and may contribute to susceptibility to emotional dysregulation in aged individuals. The present experiments used a model of chronic circadian arrhythmia generated noninvasively, via a series of circadian-disruptive light treatments, to investigate interactions between circadian desynchrony and aging on depressive- and anxiety-like behaviors, and on limbic neuroinflammatory gene expression that has been linked with emotionality. We also examined whether a social manipulation (group housing) would attenuate effects of arrhythmia on emotionality. In aged (14-18 months of age) male Siberian hamsters, circadian arrhythmia increased behavioral despair and decreased social motivation, but decreased exploratory anxiety. These effects were not evident in younger (5-9 months of age) hamsters. Social housing (3-5 hamsters/cage) abolished the effects of circadian arrhythmia on emotionality. Circadian arrhythmia alone was without effect on hippocampal or cortical interleukin-1β (IL-1β) and indoleamine 2,3-dioxygenase (Ido) mRNA expression in aged hamsters, but social housing decreased hippocampal IL-1β and Ido mRNAs. The data demonstrate that circadian disruption can negatively impact affective state, and that this effect is pronounced in older individuals. Although clear associations between circadian arrhythmia and constitutive limbic proinflammatory activity were not evident, the present data suggest that social housing markedly inhibits constitutive hippocampal IL-1β and Ido activity, which may contribute to the ameliorating effects of social housing on a number of emotional behaviors.

  14. Photoperiodic influences on ultradian rhythms of male Siberian hamsters.

    PubMed

    Prendergast, Brian J; Zucker, Irving

    2012-01-01

    Seasonal changes in mammalian physiology and behavior are proximately controlled by the annual variation in day length. Long summer and short winter day lengths markedly alter the amplitude of endogenous circadian rhythms and may affect ultradian oscillations, but the threshold photoperiods for inducing these changes are not known. We assessed the effects of short and intermediate day lengths and changes in reproductive physiology on circadian and ultradian rhythms of locomotor activity in Siberian hamsters. Males were maintained in a long photoperiod from birth (15 h light/day; 15 L) and transferred in adulthood to 1 of 7 experimental photoperiods ranging from 14 L to 9 L. Decreases in circadian rhythm (CR) robustness, mesor and amplitude were evident in photoperiods ≤14 L, as were delays in the timing of CR acrophase and expansion of nocturnal activity duration. Nocturnal ultradian rhythms (URs) were comparably prevalent in all day lengths, but 15 L markedly inhibited the expression of light-phase URs. The period (τ'), amplitude and complexity of URs increased in day lengths ≤13 L. Among hamsters that failed to undergo gonadal regression in short day lengths (nonresponders), τ' of the dark-phase UR was longer than in photoresponsive hamsters; in 13 L the incidence and amplitude of light-phase URs were greater in hamsters that did not undergo testicular regression. Day lengths as long as 14 L were sufficient to trigger changes in the waveform of CRs without affecting UR waveform. The transition from a long- to a short-day ultradian phenotype occurred for most UR components at day lengths of 12 L-13 L, thereby establishing different thresholds for CR and UR responses to day length. At the UR-threshold photoperiod of 13 L, differences in gonadal status were largely without effect on most UR parameters.

  15. Translocation and Endocytosis for Cell-penetrating Peptide Internalization

    PubMed Central

    Jiao, Chen-Yu; Delaroche, Diane; Burlina, Fabienne; Alves, Isabel D.; Chassaing, Gérard; Sagan, Sandrine

    2009-01-01

    Cell-penetrating peptides (CPPs) share the property of cellular internalization. The question of how these peptides reach the cytoplasm of cells is still widely debated. Herein, we have used a mass spectrometry-based method that enables quantification of internalized and membrane-bound peptides. Internalization of the most used CPP was studied at 37 °C (endocytosis and translocation) and 4 °C (translocation) in wild type and proteoglycan-deficient Chinese hamster ovary cells. Both translocation and endocytosis are internalization pathways used by CPP. The choice of one pathway versus the other depends on the peptide sequence (not the number of positive changes), the extracellular peptide concentration, and the membrane components. There is no relationship between the high affinity of these peptides for the cell membrane and their internalization efficacy. Translocation occurs at low extracellular peptide concentration, whereas endocytosis, a saturable and cooperative phenomenon, is activated at higher concentrations. Translocation operates in a narrow time window, which implies a specific lipid/peptide co-import in cells. PMID:19833724

  16. Magnetically-Guided Penetrant Applicator

    NASA Technical Reports Server (NTRS)

    Molina, Orlando G.

    1990-01-01

    Small wheeled vehicle moved inside nonmagnetic enclosure. Miniature magnetically guided truck uses foam-rubber sponge pads to apply penetrant fluid for inspection of welds in hidden surfaces of nonmagnetic tubes. Risk of explosion less than if electric motor used to drive vehicle. Inexpensive to make and made in range of sizes.

  17. Simulation of laser penetration efficiency

    NASA Astrophysics Data System (ADS)

    Semak, V. V.; Miller, T. F.

    2013-09-01

    The results of numerical simulation of laser beam interaction with a hypothetical metallic material with properties similar to a steel alloy are reported. The numerical simulation was performed using a physical model that includes detailed consideration of surface evaporation, evaporative cooling of the surface and evaporation recoil induced melt ejection. The laser beam ‘penetration’ is considered in terms of melting through the sample or drilling through the sample due to both evaporation and recoil ejection of material. As a demonstration of the predictive capabilities of the model, the average velocity of penetration through a material with steel-like properties is numerically predicted for various laser interaction parameters such as, laser beam radius, laser pulse duration (including CW regime), laser pulse energy and pulse repetition. In particular, the average penetration velocities through a sample due to melting are compared for pulsed and CW lasers of the same power. For the sake of another demonstration of penetration simulation, the temporal dynamics of the position of melt front relative to the sample surface irradiated by a laser beam was computed for different laser pulse repetition rates and constant average laser power. An illustration of the penetration efficiency (W parameter) defined as the amount of energy per unit volume delivered into a target in order to achieve either melting of drilling through a target wall is shown in a wide range of laser pulse parameters covering regimes corresponding to domination of melting through and drilling through.

  18. FAA Fluorescent Penetrant Laboratory Inspections

    SciTech Connect

    WINDES,CONNOR L.; MOORE,DAVID G.

    2000-08-02

    The Federal Aviation Administration Airworthiness Assurance NDI Validation Center currently assesses the capability of various non-destructive inspection (NDI) methods used for analyzing aircraft components. The focus of one such exercise is to evaluate the sensitivity of fluorescent liquid penetrant inspection. A baseline procedure using the water-washable fluorescent penetrant method defines a foundation for comparing the brightness of low cycle fatigue cracks in titanium test panels. The analysis of deviations in the baseline procedure will determine an acceptable range of operation for the steps in the inspection process. The data also gives insight into the depth of each crack and which step(s) of the inspection process most affect penetrant sensitivities. A set of six low cycle fatigue cracks produced in 6.35-mm thick Ti-6Al-4V specimens was used to conduct the experiments to produce sensitivity data. The results will document the consistency of the crack readings and compare previous experiments to find the best parameters for water-washable penetrant.

  19. In dystrophic hamsters losartan affects control of ventilation and dopamine D1 receptor density.

    PubMed

    Schlenker, Evelyn H

    2010-08-31

    The BIO 14.6 hamster (DV), an animal model of limb-girdle muscular dystrophy, has elevated angiotensin AT1 receptors that may affect ventilation. Moreover, AT1 receptors may modulate expression of dopamine D1 receptors. We investigated if chronic treatment of BIO 14.6 hamsters (DL) with losartan, an AT1 receptor blocker, affects D1 receptor density in the striatum and nucleus tractus solitarius (NTS) and normalizes ventilation during exposure to air, hypoxia, following hypoxia, and hypercapnia, Ventilation was evaluated using plethysmography. Compared to the golden Syrian hamsters (GS), DV hamsters exhibited lower hypercapnic and hypoxic responsiveness and ventilation during hypercapnic exposure. Relative to GS, DL hamsters increased breathing frequency in air and maintained ventilation during hypercapnia. Post-hypoxic minute ventilation decline occurred in DV but not in DL or GS hamsters. DL hamsters exhibited higher D1 receptor density in the striatum and NTS relative to DV hamsters. Thus, in dystrophic hamsters chronic losartan treatment stimulated frequency of breathing and increased the density of D1 receptors.

  20. [Features of infection in hamsters by Junín virus].

    PubMed

    Contigiani, M S; Sabattini, M S

    1983-01-01

    Suckling hamsters infected with three low passaged strains of Junin virus by intracerebral route developed a non differentiated illness with about 7 log of virus replication in the brain after which they either died between 6 and 19 days post inoculation or developed CF antibodies. Even in suckling hamsters and suckling mice gave similar results for viral titration, isolation attempts were less successful in hamsters. The young hamster response was characterized by a severe neurological disease and subsequent recovery with the development of CF antibodies 3). Except for isolation efficiency, the results obtained with Junin virus are similar to those reported for other arenavirus.

  1. Food hoarding is increased by food deprivation and decreased by leptin treatment in Syrian hamsters.

    PubMed

    Buckley, Carolyn A; Schneider, Jill E

    2003-11-01

    Compensatory increases in food intake are commonly observed after a period of food deprivation in many species, including laboratory rats and mice. Thus it is interesting that Syrian hamsters fail to increase food intake after a period of food deprivation, despite a fall in plasma leptin concentrations similar to those seen in food-deprived rats and mice. In previous laboratory studies, food-deprived Syrian hamsters increased the amount of food hoarded. We hypothesized that leptin treatment during food deprivation would attenuate food-deprivation-induced increases in hoarding. Baseline levels of hoarding were bimodally distributed, with no hamsters showing intermediate levels of hoarding. Both high (HH) and low hoarding (LH) hamsters were included in each experimental group. Fifty-six male hamsters were either food deprived or given ad libitum access to food for 48 h. One-half of each group received intraperitoneal injections of leptin (4 mg/kg) or vehicle every 12 h during the food-deprivation period. Within the HH group, the hoarding score increased significantly in food-deprived but not fed hamsters (P < 0.05). Leptin treatment significantly decreased hoarding in the food-deprived HH hamsters (P < 0.05). The LH hamsters did not increase hoarding regardless of whether they were food deprived or had ad libitum access to food. These results are consistent with the idea that HH hamsters respond to energetic challenges at least in part by changing their hoarding behavior and that leptin might be one factor that mediates this response.

  2. Melanin content of hamster tissues, human tissues, and various melanomas

    SciTech Connect

    Watts, K.P.; Fairchild, R.G.; Slatkin, D.N.; Greenberg, D.; Packer, S.; Atkins, H.L.; Hannon, S.J.

    1981-02-01

    Melanin content (percentage by weight) was determined in both pigmented and nonpigmented tissues of Syrian golden hamsters bearing Greene melanoma. Melanin content was also measured in various other melanoma models (B-16 in C57 mice, Harding-Passey in BALB/c mice, and KHDD in C3H mice) and in nine human melanomas, as well as in selected normal tissues. The purpose was to evaluate the possible efficacy of chlorpromazine, which is known to bind to melanin, as a vehicle for boron transport in neutron capture therapy. Successful therapy would depend upon selective uptake and absolute concentration of borated compounds in tumors; these parameters will in turn depend upon melanin concentration in melanomas and nonpigmented ''background'' tissues. Hamster whole eyes, hamster melanomas, and other well-pigmented animal melanomas were found to contain 0.3 to 0.8% melanin by weight, whereas human melanomas varied from 0.1 to 0.9% (average, 0.35%). Other tissues, with the exception of skin, were lower in content by a factor of greater than or equal to30. Melanin pigment was extracted from tissues, and the melanin content was determined spectrophotometrically. Measurements were found to be sensitive to the presence of other proteins. Previous procedures for isolating and quantifying melanin often neglected the importance of removing proteins and other interfering nonmelanic substances.

  3. Teratogenicity and embryotoxicity of nickel carbonyl in Syrian hamsters

    SciTech Connect

    Sunderman, F.W. Jr.; Shen, S.K.; Reid, M.C.; Allpass, P.R.

    1980-01-01

    Nickel carbonyl was administered to groups of pregnant hamsters by inhalation on days 4, 5, 6, 7, or 8 of gestation. The dams were killed on day 15 of gestation, and the fetuses were examined for malformations. Exposure to Ni(CO)/sub 4/ on days 4 or 5 of gestation resulted in malformation in 5.5% and 5.8% of the progeny, respectively. Progeny included 9 fetuses with cystic lungs, 7 fetuses with exencephaly, 1 fetus with exencephaly plus fused rib and 1 fetus with anophthalmia plus cleft palate. Hemorrhages into serious cavities were found. In progeny of dams exposed to Ni(CO)/sub 4/ on days 6 or 7 of gestation, there was 1 fetus with fused ribs and there were 2 fetuses with hydronephrosis. In another experiment, pregnant hamsters were exposed to inhalation of Ni(CO)/sub 4/ on day 5 of gestation; these dams were permitted to deliver their litters and to nurse their pups. There was no significant difference in the average number of live pups in the Ni(CO)/sub 4/-exposed litters compared to control litters. Neonatal mortality was increased in Ni(CO)/sub 4/-exposed litters. This study demonstrates that Ni(CO)/sub 4/ is teratogenic and embryotoxic in Syrian hamsters.

  4. Experimental Models in Syrian Golden Hamster Replicate Human Acute Pancreatitis

    PubMed Central

    Wang, Yunan; Kayoumu, Abudurexiti; Lu, Guotao; Xu, Pengfei; Qiu, Xu; Chen, Liye; Qi, Rong; Huang, Shouxiong; Li, Weiqin; Wang, Yuhui; Liu, George

    2016-01-01

    The hamster has been shown to share a variety of metabolic similarities with humans. To replicate human acute pancreatitis with hamsters, we comparatively studied the efficacy of common methods, such as the peritoneal injections of caerulein, L-arginine, the retrograde infusion of sodium taurocholate, and another novel model with concomitant administration of ethanol and fatty acid. The severity of pancreatitis was evaluated by serum amylase activity, pathological scores, myeloperoxidase activity, and the expression of inflammation factors in pancreas. The results support that the severity of pathological injury is consistent with the pancreatitis induced in mice and rat using the same methods. Specifically, caerulein induced mild edematous pancreatitis accompanied by minimal lung injury, while L-arginine induced extremely severe pancreatic injury including necrosis and neutrophil infiltration. Infusion of Na-taurocholate into the pancreatic duct induced necrotizing pancreatitis in the head of pancreas and lighter inflammation in the distal region. The severity of acute pancreatitis induced by combination of ethanol and fatty acids was between the extent of caerulein and L-arginine induction, with obvious inflammatory cells infiltration. In view of the advantages in lipid metabolism features, hamster models are ideally suited for the studies of pancreatitis associated with altered metabolism in humans. PMID:27302647

  5. Lack of carcinogenicity of cadmium chloride in female Syrian hamsters.

    PubMed

    Waalkes, M P; Rehm, S

    1998-04-01

    Cadmium is very effective at inducing necrosis within the ovaries of rodents, and the Syrian hamster appears particularly sensitive. The extent of cadmium-induced necrosis depends on the stage of the estrous cycle and is most pronounced when injected on the day prior to ovulation (proestrous). In male rodents cadmium induces a similar necrosis within the testes, which given sufficient time can lead to the development of testicular tumors. In this study we tested the hypothesis that cadmium-induced ovarian necrosis could eventually lead to tumor formation. In sexually mature groups of female Syrian hamsters (> 8 weeks old; n = 50-59), the estrous cycle was determined by visual inspection of vaginal discharge for four consecutive cycles. The animals were then given cadmium (0, 30, 40 and 50 micromol/kg) subcutaneously as a single injection in the dorsal thoracic midline on cycle day 4 (proestrous). Based on prior work, these doses are sufficient to induce extensive acute ovarian damage. Animals were then observed over the next 78 weeks. Although survival and body weight were reduced by cadmium, treatment with the metal did not result in an enhanced incidence of tumors at any site including the ovaries. Non-neoplastic lesions such as amyloidosis and pancreatic hepatocytes were linked to cadmium exposure. These results indicate that the association of cadmium-induced testicular necrosis with tumor development seen in males does not occur in the Syrian hamster ovaries. PMID:9674965

  6. Experimental Models in Syrian Golden Hamster Replicate Human Acute Pancreatitis.

    PubMed

    Wang, Yunan; Kayoumu, Abudurexiti; Lu, Guotao; Xu, Pengfei; Qiu, Xu; Chen, Liye; Qi, Rong; Huang, Shouxiong; Li, Weiqin; Wang, Yuhui; Liu, George

    2016-01-01

    The hamster has been shown to share a variety of metabolic similarities with humans. To replicate human acute pancreatitis with hamsters, we comparatively studied the efficacy of common methods, such as the peritoneal injections of caerulein, L-arginine, the retrograde infusion of sodium taurocholate, and another novel model with concomitant administration of ethanol and fatty acid. The severity of pancreatitis was evaluated by serum amylase activity, pathological scores, myeloperoxidase activity, and the expression of inflammation factors in pancreas. The results support that the severity of pathological injury is consistent with the pancreatitis induced in mice and rat using the same methods. Specifically, caerulein induced mild edematous pancreatitis accompanied by minimal lung injury, while L-arginine induced extremely severe pancreatic injury including necrosis and neutrophil infiltration. Infusion of Na-taurocholate into the pancreatic duct induced necrotizing pancreatitis in the head of pancreas and lighter inflammation in the distal region. The severity of acute pancreatitis induced by combination of ethanol and fatty acids was between the extent of caerulein and L-arginine induction, with obvious inflammatory cells infiltration. In view of the advantages in lipid metabolism features, hamster models are ideally suited for the studies of pancreatitis associated with altered metabolism in humans. PMID:27302647

  7. Learned magnetic compass orientation by the Siberian hamster, Phodopus sungorus

    SciTech Connect

    Deutschlander, Mark E.; Freake, Michael J.; Borland, Christopher; Phillips, John B.; Madden, R C.; Anderson, Larry E.; Wilson, B W.

    2003-04-01

    Magnetic orientation has been demonstrated in Siberian hamsters, Phodopus sungorus. The behavior, using a nest building assay, shows a directional preference in nest position and appears in this animal to be a learned behavior. Hamsters were housed prior to testing in rectangular cages aligned along perpendicular axes. When subsequently tested in a radially-symmetrical arena, the hamsters positioned their nests in a bimodal distribution that coincided with the magnetic direction of the long-axis of the holding cages. In addition, results are presented that illustrate some of the factors that can influence behavioral responses to the magnetic field. In particular for P. sungorus, holding conditions prior to testing and the presence of non-magnetic cues may influence the strength and expression of magnetic orientation. Failure to consider these and other factors may help to explain why previous attempts to demonstrate magnetic orientation in a number of rodent species have failed or, when positive results have been obtained, have been difficult to replicate in other laboratories.

  8. Female-biased anorexia and anxiety in the Syrian hamster.

    PubMed

    Shannonhouse, John L; Fong, Li An; Clossen, Bryan L; Hairgrove, Ross E; York, Daniel C; Walker, Benjamin B; Hercules, Gregory W; Mertesdorf, Lauren M; Patel, Margi; Morgan, Caurnel

    2014-06-22

    Anorexia and anxiety cause significant mortality and disability with female biases and frequent comorbidity after puberty, but the scarcity of suitable animal models impedes understanding of their biological underpinnings. It is reported here that in adult or weanling Syrian hamsters, relative to social housing (SH), social separation (SS) induced anorexia characterized as hypophagia, weight loss, reduced adiposity, and hypermetabolism. Following anorexia, SS increased reluctance to feed, and thigmotaxis, in anxiogenic environments. Importantly, anorexia and anxiety were induced post-puberty with female biases. SS also reduced hypothalamic corticotrophin-releasing factor mRNA and serum corticosteroid levels assessed by RT-PCR and RIA, respectively. Consistent with the view that sex differences in adrenal suppression contributed to female biases in anorexia and anxiety by disinhibiting neuroimmune activity, SS elevated hypothalamic interleukin-6 and toll-like receptor 4 mRNA levels. Although corticosteroids were highest during SH, they were within the physiological range and associated with juvenile-like growth of white adipose, bone, and skeletal muscle. These results suggest that hamsters exhibit plasticity in bioenergetic and emotional phenotypes across puberty without an increase in stress responsiveness. Thus, social separation of hamsters provides a model of sex differences in anorexia and anxiety during adulthood and their pathogeneses during adolescence.

  9. Percutaneous penetration--methodological considerations.

    PubMed

    Holmgaard, Rikke; Benfeldt, Eva; Nielsen, Jesper B

    2014-07-01

    Studies on percutaneous penetration are needed to assess the hazards after unintended occupational skin exposures to industrial products as well as the efficacy after intended consumer exposure to topically applied medicinal or cosmetic products. During recent decades, a number of methods have been developed to replace methods involving experimental animals. The results obtained from these methods are decided not only by the chemical or product tested, but to a significant degree also by the experimental set-up and decisions made by the investigator during the planning phase. The present MiniReview discusses some of the existing and well-known experimental in vitro and in vivo methods for studies of percutaneous penetration together with some more recent and promising methods. After this, some considerations and recommendations about advantages and limitations of the different methods and their relevance for the prediction of percutaneous penetration are given. Which method to prefer will depend on the product to be tested and the question asked. Regulatory guidelines exist for studies on percutaneous penetration, but researchers as well as regulatory bodies need to pay specific attention to the vehicles and solvents used in donor and sampling fluids so that it reflects in-use conditions as closely as possible. Based on available experimental data, mathematical models have been developed to aid predictions of skin penetration. The authors question the general use of the present mathematical models in hazard assessment, as they seem to ignore outliers among chemicals as well as the heterogeneity of skin barrier properties and skin conditions within the exposed populations. PMID:24373389

  10. Vitrification of oocytes from endangered Mexican gray wolves (Canis lupus baileyi).

    PubMed

    Boutelle, S; Lenahan, K; Krisher, R; Bauman, K L; Asa, C S; Silber, S

    2011-03-01

    Careful genetic management, including cryopreservation of genetic material, is central to conservation of the endangered Mexican gray wolf. We tested a technique, previously used to vitrify human and domestic animal oocytes, on oocytes from domestic dogs as a model and from the endangered Mexican wolf. This method provided a way to conserve oocytes from genetically valuable older female Mexican wolves as an alternative to embryos for preserving female genes. Oocytes were aspirated from ovaries of 36 female dogs in December and March (0 to 65 oocytes per female) and from six female wolves (4 to 73 per female) during their physiologic breeding season, or following stimulation with the GnRH agonist deslorelin. Oocytes from dogs were pooled; half were immediately tested for viability and the remainder vitrified, then warmed and tested for viability. All oocytes were vitrified by being moved through media of increasing cryoprotectant concentration, placed on Cryotops, and plunged into liquid nitrogen. There was no difference in viability (propidium iodide staining) between fresh and vitrified, warmed dog oocytes (65.7 and 61.0%, respectively, P = 0.27). Oocyte viability after warming was similarly assessed in a subset of wolves (4 to 15 oocytes from each of three females; total 29 oocytes). Of these, 57.1% of the post-thaw intact oocytes were viable, which was 41.4% of all oocytes warmed. These were the first oocytes from a canid or an endangered species demonstrated to have maintained viability after vitrification and warming. Furthermore, our results demonstrated that vitrification of oocytes with the Cryotop technique was an option for preserving female gametes from Mexican wolves for future use in captive breeding programs, although in vitro embryo production techniques must first be developed in canids for this technique to be used.

  11. The fertilization ability and developmental competence of bovine oocytes grown in vitro.

    PubMed

    Makita, Miho; Ueda, Mayuko; Miyano, Takashi

    2016-08-25

    In vitro growth culture systems for oocytes are being developed in several mammalian species. In these growth culture systems, in vitro grown oocytes usually have lower blastocyst formation than in vivo grown oocytes after in vitro fertilization. Furthermore, there have been a few reports that investigated the fertilization ability of in vitro grown oocytes in large animals. The purpose of this study was to investigate the fertilization process and developmental competence of bovine oocytes grown in vitro. Oocyte-granulosa cell complexes collected from bovine early antral follicles (0.4-0.7 mm in diameter) were cultured for growth with 17β-estradiol and androstenedione for 14 days and matured in vitro. These oocytes were then inseminated for 6 or 12 h, and further cultured for development up to 8 days in vitro. After growth culture, oocytes grew from 95 µm to around 120 µm and acquired maturation competence (79%). Although fertilization rates of in vitro grown oocytes were low after 6 h of insemination, 34% of in vitro grown oocytes fertilized normally after 12 h of insemination, having two polar bodies and two pronuclei with a sperm tail, and 22% of these oocytes developed into blastocysts after 8 days of culture. The fertilization and blastocyst formation rates were similar to those of in vivo grown oocytes. In addition, blastocyst cell numbers were also similar between in vitro and in vivo grown oocytes. In conclusion, in vitro grown bovine oocytes are similar to in vivo grown oocytes in fertilization ability and can develop into blastocysts.

  12. RNA-Seq Profiling of Intact and Enucleated Oocyte SCNT Embryos Reveals the Role of Pig Oocyte Nucleus in Somatic Reprogramming

    PubMed Central

    Bai, Lin; Li, Mengqi; Sun, Junli; Yang, Xiaogan; Lu, Yangqing; Lu, Shengsheng; Lu, Kehuan

    2016-01-01

    The specific molecular mechanisms involved in somatic reprogramming remain unidentified. Removal of the oocyte genome is one of the primary causes of developmental failure in cloned embryos, whereas intact oocyte shows stronger reprogramming capability than enucleated oocyte. To identify the reason for the low efficiency of cloning and elucidate the mechanisms involved in somatic reprogramming by the oocyte nucleus, we injected pig cumulus cells into 539 intact MII oocytes and 461 enucleated MII oocytes. Following activation, 260 polyploidy embryos developed to the blastocyst stage whereas only 93 traditionally cloned embryos (48.2% vs. 20.2%, P < 0.01) reached blastocyst stage. Blastocysts generated from intact oocytes also had more cells than those generated from enucleated oocytes (60.70 vs. 46.65, P < 0.01). To identify the genes that contribute to this phenomenon, two early embryos in 2-cell and 4-cell stages were collected for single-cell RNA sequencing. The two kinds of embryos were found to have dramatically different transcriptome profiles. Intact oocyte nuclear transfer embryos showed 1,738 transcripts that were up-regulated relative to enucleated cloned embryos at the 2-cell stage and 728 transcripts that were down-regulated (|log2Ratio| ≥ 5). They showed 2,941 transcripts that were up-regulated during the 4-cell stage and 1,682 that were down-regulated (|log2Ratio| ≥ 5). The most significantly enriched gene ontology categories were those involved in the regulation of binding, catalytic activity, and molecular transducer activity. Other genes that were notably up-regulated and expressed in intact oocyte nuclear transfer embryos were metabolic process. This study provides a comprehensive profile of the differences in gene expression between intact oocyte nuclear transfer embryos and traditional nuclear transfer embryos. This work thus paves the way for further research on the mechanisms underlying somatic reprogramming by oocytes. PMID:27070804

  13. RNA-Seq Profiling of Intact and Enucleated Oocyte SCNT Embryos Reveals the Role of Pig Oocyte Nucleus in Somatic Reprogramming.

    PubMed

    Bai, Lin; Li, Mengqi; Sun, Junli; Yang, Xiaogan; Lu, Yangqing; Lu, Shengsheng; Lu, Kehuan

    2016-01-01

    The specific molecular mechanisms involved in somatic reprogramming remain unidentified. Removal of the oocyte genome is one of the primary causes of developmental failure in cloned embryos, whereas intact oocyte shows stronger reprogramming capability than enucleated oocyte. To identify the reason for the low efficiency of cloning and elucidate the mechanisms involved in somatic reprogramming by the oocyte nucleus, we injected pig cumulus cells into 539 intact MII oocytes and 461 enucleated MII oocytes. Following activation, 260 polyploidy embryos developed to the blastocyst stage whereas only 93 traditionally cloned embryos (48.2% vs. 20.2%, P < 0.01) reached blastocyst stage. Blastocysts generated from intact oocytes also had more cells than those generated from enucleated oocytes (60.70 vs. 46.65, P < 0.01). To identify the genes that contribute to this phenomenon, two early embryos in 2-cell and 4-cell stages were collected for single-cell RNA sequencing. The two kinds of embryos were found to have dramatically different transcriptome profiles. Intact oocyte nuclear transfer embryos showed 1,738 transcripts that were up-regulated relative to enucleated cloned embryos at the 2-cell stage and 728 transcripts that were down-regulated (|log2Ratio| ≥ 5). They showed 2,941 transcripts that were up-regulated during the 4-cell stage and 1,682 that were down-regulated (|log2Ratio| ≥ 5). The most significantly enriched gene ontology categories were those involved in the regulation of binding, catalytic activity, and molecular transducer activity. Other genes that were notably up-regulated and expressed in intact oocyte nuclear transfer embryos were metabolic process. This study provides a comprehensive profile of the differences in gene expression between intact oocyte nuclear transfer embryos and traditional nuclear transfer embryos. This work thus paves the way for further research on the mechanisms underlying somatic reprogramming by oocytes. PMID:27070804

  14. Chromosome Cohesion Established by Rec8-Cohesin in Fetal Oocytes Is Maintained without Detectable Turnover in Oocytes Arrested for Months in Mice

    PubMed Central

    Burkhardt, Sabrina; Borsos, Máté; Szydlowska, Anna; Godwin, Jonathan; Williams, Suzannah A.; Cohen, Paula E.; Hirota, Takayuki; Saitou, Mitinori; Tachibana-Konwalski, Kikuë

    2016-01-01

    Summary Sister chromatid cohesion mediated by the cohesin complex is essential for chromosome segregation in mitosis and meiosis [1]. Rec8-containing cohesin, bound to Smc3/Smc1α or Smc3/Smc1β, maintains bivalent cohesion in mammalian meiosis [2, 3, 4, 5, 6]. In females, meiotic DNA replication and recombination occur in fetal oocytes. After birth, oocytes arrest at the prolonged dictyate stage until recruited to grow into mature oocytes that divide at ovulation. How cohesion is maintained in arrested oocytes remains a pivotal question relevant to maternal age-related aneuploidy. Hypothetically, cohesin turnover regenerates cohesion in oocytes. Evidence for post-replicative cohesion establishment mechanism exists, in yeast and invertebrates [7, 8]. In mouse fetal oocytes, cohesin loading factor Nipbl/Scc2 localizes to chromosome axes during recombination [9, 10]. Alternatively, cohesion is maintained without turnover. Consistent with this, cohesion maintenance does not require Smc1β transcription, but unlike Rec8, Smc1β is not required for establishing bivalent cohesion [11, 12]. Rec8 maintains cohesion without turnover during weeks of oocyte growth [3]. Whether the same applies to months or decades of arrest is unknown. Here, we test whether Rec8 activated in arrested mouse oocytes builds cohesion revealed by TEV cleavage and live-cell imaging. Rec8 establishes cohesion when activated during DNA replication in fetal oocytes using tamoxifen-inducible Cre. In contrast, no new cohesion is detected when Rec8 is activated in arrested oocytes by tamoxifen despite cohesin synthesis. We conclude that cohesion established in fetal oocytes is maintained for months without detectable turnover in dictyate-arrested oocytes. This implies that women’s fertility depends on the longevity of cohesin proteins that established cohesion in utero. PMID:26898469

  15. Androgen dependence in hamsters: overdose, tolerance, and potential opioidergic mechanisms.

    PubMed

    Peters, K D; Wood, R I

    2005-01-01

    Anabolic steroids are drugs of abuse. However, the potential for steroid reward and addiction remains largely unexplored. This study used i.c.v. testosterone self-administration and controlled infusions of testosterone or vehicle in hamsters to explore central mechanisms of androgen overdose. Forty-two hamsters used nose-pokes to self-administer 1 microg/microl testosterone i.c.v. 4 h/day in an operant chamber. During 1-56 days of androgen self-administration, 10 (24%) hamsters died. Deaths correlated with peak daily intake of testosterone. Of the hamsters that self-administered a peak intake of <20 microg/day, there was 100% survival (10/10). Survival decreased to 86% (19/22) when daily testosterone intake peaked at 20-60 microg/day. Only 30% (three of 10) survived when daily testosterone intake exceeded 60 microg/day. Deaths are not due to volume or vehicle because i.c.v. infusions of 80 mul vehicle had no effect. Testosterone overdose resembles opiate intoxication. When male hamsters received infusions of 40 microg testosterone, locomotion (25.1+/-18.8 grid-crossings/10 min), respiration (72.7+/-5.4 breaths/min) and body temperature (33.5+/-0.4 degrees C) were significantly reduced, compared with males receiving vehicle infusions (186.1+/-8.1 crossings/10 min, 117.6+/-1.0 breaths/min, 35.9+/-0.1 degrees C, P<0.05). However, males developed tolerance to continued daily testosterone infusion. After 15 days, locomotion (170.2+/-6.3 crossings), respiration (118.4+/-1.3 breaths/min), and body temperature (35.3+/-0.3 degrees C) in testosterone-infused males were equivalent to that in vehicle controls (P>0.05). The depressive effects of testosterone infusion are blocked by the opioid antagonist, naltrexone. With naltrexone pre-treatment (10 mg/kg s.c.), locomotion (183.7+/-1.8 crossings/10 min), respiration (116.9+/-0.3 breaths/min), and body temperature (36.1+/-0.4 degrees C) during testosterone infusion were equivalent to vehicle controls. Likewise, naltrexone

  16. Cytoskeleton and Cytoskeleton-Bound RNA Visualization in Frog and Insect Oocytes.

    PubMed

    Kloc, Malgorzata; Bilinski, Szczepan; Kubiak, Jacek Z

    2016-01-01

    The majority of oocyte functions involves and depends on the cytoskeletal elements, which include microtubules and actin and cytokeratin filaments. Various structures and molecules are temporarily or permanently bound to the cytoskeletal elements and their functions rely on cytoskeleton integrity and its timely assembly. Thus the accurate visualization of cytoskeleton is often crucial for studies and analyses of oocyte structure and functions. Here we describe several reliable methods for microtubule and/or microfilaments preservation and visualization in Xenopus oocyte extracts, and in situ in live and fixed insect and frog (Xenopus) oocytes. In addition, we describe visualization of cytoskeleton-bound RNAs using molecular beacons in live Xenopus oocytes. PMID:27557581

  17. Cytoskeleton and Cytoskeleton-Bound RNA Visualization in Frog and Insect Oocytes.

    PubMed

    Kloc, Malgorzata; Bilinski, Szczepan; Kubiak, Jacek Z

    2016-01-01

    The majority of oocyte functions involves and depends on the cytoskeletal elements, which include microtubules and actin and cytokeratin filaments. Various structures and molecules are temporarily or permanently bound to the cytoskeletal elements and their functions rely on cytoskeleton integrity and its timely assembly. Thus the accurate visualization of cytoskeleton is often crucial for studies and analyses of oocyte structure and functions. Here we describe several reliable methods for microtubule and/or microfilaments preservation and visualization in Xenopus oocyte extracts, and in situ in live and fixed insect and frog (Xenopus) oocytes. In addition, we describe visualization of cytoskeleton-bound RNAs using molecular beacons in live Xenopus oocytes.

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

    PubMed

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

    2015-02-01

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

  19. A simplified approach for oocyte enucleation in mammalian cloning.

    PubMed

    Iuso, Domenico; Czernik, Marta; Zacchini, Federica; Ptak, Grazyna; Loi, Pasqualino

    2013-12-01

    Despite its success in almost all farm and laboratory animals, somatic cell nuclear transfer (SCNT) is still a low-efficiency technique. In this investigation, we determined the impact of each enucleation step on oocyte viability (assessed by parthenogenetic activation): Hoechst (HO) staining, cytochalasin B, ultraviolet (UV) exposure, and demecolcine. Our data showed that of all the factors analyzed, UV exposure impaired oocyte development (cleavage, 59% for untreated oocytes vs. 8% UV exposed; blastocyst stage, 32% untreated vs. 0% UV exposed). A minor toxicity was detected following demecolcine treatment (cleavage, 62%; blastocyst stage, 13%). Next, we compared HO/UV (canonical) and demecolcine-assisted enucleation (DAE), with a straight removal of metaphase chromosomes without any chemical or physical aid (straight enucleation). DAE improved the preimplantation development of sheep cloned embryos compared to HO/UV enucleation (cleavage, 38% vs. 19%; blastocysts, 17% vs. 4%), yet straight enucleation resulted in the highest cleavage and blastocysts rates (61% and 30%, respectively). We concluded that: (1) UV exposure harms sheep oocyte and embryo development; (2) DAE may represent an alternative approach, especially for unskilled operators; and (3) straight enucleation remains, in our estimation, the most reliable and least harmful protocol for SCNT.

  20. Protein tyrosine phosphorylation during meiotic divisions of starfish oocytes

    SciTech Connect

    Peaucellier, G.; Andersen, A.C.; Kinsey, W.H. )

    1990-04-01

    We have used an antibody specific for phosphotyrosine to investigate protein phosphorylation on tyrosine during hormone-induced maturation of starfish oocytes. Analysis of immunoprecipitates from cortices of in vivo labeled Marthasterias glacialis oocytes revealed the presence of labeled phosphotyrosine-containing proteins only after hormone addition. Six major phosphoproteins of 195, 155, 100, 85, 45, and 35 kDa were detected. Total activity in immunoprecipitates increased until first polar body emission and was greatly reduced upon completion of meiosis but some proteins exhibited different kinetics. The labeling of the 155-kDa protein reached a maximum at germinal vesicle breakdown, while the 35-kDa appeared later and disappeared after polar body emission. Similar results were obtained with Asterias rubens oocytes. In vitro phosphorylation of cortices showed that tyrosine kinase activity is a major protein kinase activity in this fraction, the main endogenous substrate being a 68-kDa protein. The proteins phosphorylated on tyrosine in vitro were almost similar in extracts from oocytes treated or not with the hormone.

  1. The role of progesterone in oocyte acquisition of developmental competence.

    PubMed

    Fair, T; Lonergan, P

    2012-08-01

    It is generally accepted that progesterone (P4) is a key regulator of reproductive function in mammals. In cattle, the primary focus of P4's actions has been uterine receptivity and maintenance of pregnancy. Studies in mammalian laboratory species and ovarian derived cell lines also highlight their role in ovarian function. Extensive research in non-mammalian species has elucidated a critical role for P4 and both its nuclear and membrane-bound receptors in oocyte maturation and ovulation. Until recently, such a role in mammalian oocytes has been disputed. However, as oestrous synchronization regimes are constantly tweaked and revised to maximize pregnancy rates to artificial insemination in cattle, the importance of P4 priming of the dominant ingfollicle is once again tak centre stage. Sequencing of the bovine genome and the development of multiple transcriptomic data mining tools have facilitated an explosion in global transcriptome profiling of immature and matured oocytes and their surrounding cumulus cells. Many of the differentially regulated genes and their associated preferentially populated pathways appear to be P4 regulated in other tissues. Therefore, attention is once again turning to a potential role for P4 in ovulatory follicle development and oocyte maturation in cattle. The current review summarizes the most recent findings in these areas. PMID:22827363

  2. Ribosome crystals in the oocyte of Gerris najas (Heteroptera).

    PubMed

    Choi, W C; Nagl, W

    1977-01-01

    Oocytes of the pond skater, Gerris najas, display ribosome tetramers that are arranged in the form of sheets in the vicinity of the nucleus. This is the first finding of ribosome crystals in an insect and suggests that ribosome crystallization may be a common phenomenon of cells that are inactive in protein synthesis.

  3. Fourteen babies born after round spermatid injection into human oocytes

    PubMed Central

    Tanaka, Atsushi; Nagayoshi, Motoi; Takemoto, Youichi; Tanaka, Izumi; Kusunoki, Hiroshi; Watanabe, Seiji; Kuroda, Keiji; Takeda, Satoru; Ito, Masahiko; Yanagimachi, Ryuzo

    2015-01-01

    During the human in vitro fertilization procedure in the assisted reproductive technology, intracytoplasmic sperm injection is routinely used to inject a spermatozoon or a less mature elongating spermatid into the oocyte. In some infertile men, round spermatids (haploid male germ cells that have completed meiosis) are the most mature cells visible during testicular biopsy. The microsurgical injection of a round spermatid into an oocyte as a substitute is commonly referred to as round spermatid injection (ROSI). Currently, human ROSI is considered a very inefficient procedure and of no clinical value. Herein, we report the birth and development of 14 children born to 12 women following ROSI of 734 oocytes previously activated by an electric current. The round spermatids came from men who had been diagnosed as not having spermatozoa or elongated spermatids by andrologists at other hospitals after a first Micro-TESE. A key to our success was our ability to identify round spermatids accurately before oocyte injection. As of today, all children born after ROSI in our clinic are without any unusual physical, mental, or epigenetic problems. Thus, for men whose germ cells are unable to develop beyond the round spermatid stage, ROSI can, as a last resort, enable them to have their own genetic offspring. PMID:26575628

  4. Akon - A Penetrator for Europa

    NASA Astrophysics Data System (ADS)

    Jones, Geraint

    2016-04-01

    Jupiter's moon Europa is one of the most intriguing objects in our Solar System. This 2000km-wide body has a geologically young solid water ice crust that is believed to cover a global ocean of liquid water. The presence of this ocean, together with a source of heating through tidal forces, make Europa a conceivable location for extraterrestrial life. The science case for exploring all aspects of this icy world is compelling. NASA has selected the Europa Mission (formerly Europa Clipper) to study Europa in detail in the 2020s through multiple flybys, and ESA's JUICE mission will perform two flybys of the body in the 2030s. The US agency has extended to the European Space Agency an invitation to provide a contribution to their mission. European scientists interested in Europa science and exploration are currently organizing themselves, in the framework of a coordinated Europa M5 Inititative to study concurrently the main options for this ESA contribution, from a simple addition of individual instruments to the NASA spacecraft, to a lander to investigate Europa's surface in situ. A high speed lander - a penetrator - is by far the most promising technology to achieve this latter option within the anticipated mass constraints, and studies of such a hard lander, many funded by ESA, are now at an advanced level. An international team to formally propose an Europa penetrator to ESA in response to the anticipated ESA M5 call is growing. The working title of this proposal is Akon (Άκων), named after the highly accurate javelin gifted to Europa by Zeus in ancient Greek mythology. We present plans for the Akon penetrator, which would impact Europa's surface at several hundred metres per second, and travel up to several metres into the moon's subsurface. To achieve this, the penetrator would be delivered to the surface by a dedicated descent module, to be destroyed on impact following release of the penetrator above the surface. It is planned that the instruments to be

  5. Embryo apoptosis identification: Oocyte grade or cleavage stage?

    PubMed Central

    Bakri, Noraina Mohd; Ibrahim, Siti Fatimah; Osman, Nurul Atikah; Hasan, Nurhaslina; Jaffar, Farah Hanan Fathihah; Rahman, Zulaiha Abdul; Osman, Khairul

    2015-01-01

    Apoptosis is a programed cell death that is vital for tissue homeostasis. However, embryo apoptosis had been known to be related to embryo fragmentation which should be avoided in in vitro fertilization (IVF). The purpose of this study was to evaluate the relationship of embryo apoptosis with the grade of immature oocytes and cleavage stage of in vitro produced (IVP) cattle embryos. This study consisted of 345 oocytes collected through ovary slicing. Immature oocytes were graded as A, B and C. This grading was based on cumulus cell thickness and compactness. All oocytes then underwent an in vitro maturation (IVM) procedure. An IVF was done 24 h after IVM culture. Prior to staining, stage of cleaved embryos was determined and classified as either 2, 4, 8 or >8-cell embryo stage. Apoptosis status of cleaved IVP embryos was determined by using annexin V-FITC staining technique at 48 and 72 h post insemination (hpi). Apoptosis status for each embryo was classified as either early or late. The result showed that there was no significant difference (p > 0.05) of apoptosis status among grade A, B and C embryos. All grades of oocytes showed embryo apoptosis where 1.5% late apoptosis for grade A, 4.5% and 10.4% of early and late apoptosis for grade B and grade C. Early apoptosis was not seen in grade A embryo. We also noted no significant difference (p > 0.05) of apoptosis status between 2, 4, 8 and >8-cell embryo stage. Early apoptosis was also not seen in >8-cell stage. Even though there were no differences in apoptosis expression between the three classes, the cleavage rate of grade A oocytes was significantly higher (p < 0.01) than grade B and grade C. In conclusion, the apoptosis expression in the embryo can occur regardless of the oocyte quality and the cleavage stage of the embryo produced. PMID:26858565

  6. Granulosa cell-oocyte interactions: the phosphorylation of specific proteins in mouse oocytes at the germinal vesicle stage is dependent upon the differentiative state of companion somatic cells

    SciTech Connect

    Cecconi, S.; Tatone, C.; Buccione, R.; Mangia, F.; Colonna, R. )

    1991-05-01

    The role of granulosa cells in the regulation of mouse ovarian oocyte metabolism was investigated. Fully grown antral oocytes, isolated from surrounding cumulus cells, were cultured on monolayers of preantral granulosa cells in the presence of dbcAMP to prevent the resumption of meiosis. Under these conditions metabolic cooperativity was established between the two cell types as early as 1 hr after seeding. Moreover, cocultured oocytes phosphorylated two polypeptides of 74 and 21 kDa which are normally phosphorylated in follicle-enclosed growing oocytes but not in cumulus cell-enclosed fully grown oocytes at the germinal vesicle stage. When cocultured oocytes were allowed to resume meiosis, the 74 and 21 kDa proteins were synthesized but no longer phosphorylated even though intercellular coupling between the two cell types was maintained during radiolabeling. It appears therefore: (a) that the different protein kinase activity of growing and fully grown germinal vesicle-stage mouse oocytes is related to the differentiative state of granulosa cells, and (b) that the regulation of oocyte protein phosphorylation activity by granulosa cells is dependent on the meiotic stage of the oocyte.

  7. Dynamics of intracellular phospholipid membrane organization during oocyte maturation and successful vitrification of immature oocytes retrieved by ovum pick-up in cattle.

    PubMed

    Aono, Akira; Nagatomo, Hiroaki; Takuma, Tetsuya; Nonaka, Rika; Ono, Yoshitaka; Wada, Yasuhiko; Abe, Yasuyuki; Takahashi, Masashi; Watanabe, Tomomasa; Kawahara, Manabu

    2013-05-01

    The objective was to determine if immature bovine oocytes with cumulus cells at the germinal vesicle (GV) stage could be vitrified by aluminum sheets (AS; pieces of sheet-like aluminum foil). Cleavage rates in fertilized oocytes previously vitrified by the AS procedure were higher than those vitrified by a nylon-mesh holder (NM) procedure (89.3 ± 2.1% vs. 65.0 ± 3.7%). Cleaved embryos derived from the AS but not from the NM procedures developed to blastocysts. Furthermore, to investigate the effects of vitrifying GV oocytes on cytoplasmic structure and on the ability to undergo cytoplasmic changes, the intracellular phospholipid membrane (IM) was stained with the lipophilic fluorescent dye, 3,3'-dioctadecyloxa-carbocyanine perchlorate. After vitrification by AS, the IM remained intact relative to that of oocytes vitrified by NM. During in vitro maturation, reorganization of the IM was also undamaged in oocytes vitrified by AS before oocyte maturation, and the IM within oocytes vitrified by the NM procedure was evidently impaired. Finally, vitrification (AS) was used for GV oocytes collected using the ovum pick-up method. A bull calf was born after in vitro production and subsequent embryo transfer. The vitrification techniques described herein should facilitate generation of viable in vitro production bovine blastocysts using oocytes recovered using the ovum pick-up method.

  8. Advances in oocyte cryopreservation technology will eventually blur the ethical and moral boundaries between compensated egg sharing and commercialized oocyte donation.

    PubMed

    Heng, Boon Chin

    2006-03-01

    Compensated egg sharing was originally conceived as a patient self-help co-operative scheme to avoid overt 'commodification' of donor oocytes and prevent doctors and medical institutions from acting as 'brokers' of donated human material. As such, egg sharing is an ethically justifiable and much-preferred alternative to commercialized oocyte donation. However, recent advances in oocyte cryopreservation technology are likely to blur the ethical and moral boundaries between compensated egg sharing and commercialized oocyte donation. The banking of cryopreserved oocytes would negate the requirement for donors and recipients in egg sharing to have co-ordinated and synchronized treatment cycles. Instead, fertility doctors and medical institutions can now offer subsidized fertility treatment upfront to any patient willing to donate a portion of her retrieved cohort of oocytes for banking and subsequent donation. Thus, more opportunity is now open for them to act as 'middle-man' to broker the transaction of oocytes between donor and recipient, which would inevitably result in overt 'commodification' of donated human material. Administrative and processing fees will de finitely be billed to prospective recipients, for banking and storage of the cryopreserved oocytes, which would mean that a direct profit can now be made from the transaction of oocytes between donor and recipient.

  9. Impact of tightly focused femtosecond laser pulses on nucleolus-like bodies of mouse GV oocyte and the ability of mouse oocytes to mature.

    PubMed

    Astafev, A A; Zalesskiy, A D; Zatsepina, O V; Kostrov, A N; Krivoharchenko, A S; Osychenko, A A; Serobyan, G A; Nadtochenko, V A

    2016-03-01

    Using femtosecond laser radiation, nucleolus-like bodies (NLBs) of mouse oocytes were locally dissected without damage to zona pellucida, cytoplasmic membrane, nuclear membrane, and nucleoplasm surrounding NLB. It was found that, after dissection of 2.7 × 10(-11) cm(3) of NLB material, which is approximately 5.2% of 10 μm NLB volume, the probability of germinal vesicle oocyte development to metaphase II stage of meiosis decreased 3-7 times compared to the non-treated oocytes. This result indicates that NLB material organization is significant for mouse oocyte maturation. PMID:27193718

  10. Timing of meiotic progression in bovine oocytes and its effect on early embryo development.

    PubMed

    Dominko, T; First, N L

    1997-08-01

    This study was designed to investigate the effect of the kinetics of nuclear maturation in bovine oocytes on early embryo development and to examine whether the time of insemination of mature oocytes affects the oocytes' ability to support events of early embryo development. The time required for completion of nuclear maturation was influenced by gonadotropins used to supplement the maturation medium. Luteinizing hormone (LH) enhanced the speed of nuclear maturation when compared to follicle-stimulating hormone (FSH). Oocytes completing their nuclear maturation early (by 16 hours after the initiation of culture) were more likely to complete the first embryonic cell cycle (78% in LH vs. 43% in FSH) and develop to the blastocyst stage (47% in LH vs. 34% in FSH). As the age of the oocytes at the time of MII arrest increased (extrusion of the polar body by 20 or 24 hours), a decrease in their ability to cleave and develop to the blastocyst stage was observed. Differences in the oocyte's ability to decondense chromatin and form pronuclei were also observed. Early maturing oocytes started forming pronuclei earlier than their later maturing counterparts. The time of insemination of mature oocytes played an equally important role. Generally, when insemination of mature oocytes was delayed for 8 hours, higher proportions of fertilized oocytes developed to advanced preimplantation stages than did the oocytes inseminated immediately after metaphase II arrest. PMID:9211431

  11. Timing of meiotic progression in bovine oocytes and its effect on early embryo development.

    PubMed

    Dominko, T; First, N L

    1997-08-01

    This study was designed to investigate the effect of the kinetics of nuclear maturation in bovine oocytes on early embryo development and to examine whether the time of insemination of mature oocytes affects the oocytes' ability to support events of early embryo development. The time required for completion of nuclear maturation was influenced by gonadotropins used to supplement the maturation medium. Luteinizing hormone (LH) enhanced the speed of nuclear maturation when compared to follicle-stimulating hormone (FSH). Oocytes completing their nuclear maturation early (by 16 hours after the initiation of culture) were more likely to complete the first embryonic cell cycle (78% in LH vs. 43% in FSH) and develop to the blastocyst stage (47% in LH vs. 34% in FSH). As the age of the oocytes at the time of MII arrest increased (extrusion of the polar body by 20 or 24 hours), a decrease in their ability to cleave and develop to the blastocyst stage was observed. Differences in the oocyte's ability to decondense chromatin and form pronuclei were also observed. Early maturing oocytes started forming pronuclei earlier than their later maturing counterparts. The time of insemination of mature oocytes played an equally important role. Generally, when insemination of mature oocytes was delayed for 8 hours, higher proportions of fertilized oocytes developed to advanced preimplantation stages than did the oocytes inseminated immediately after metaphase II arrest.

  12. The Effects of Voluntary Exercise on Oocyte Quality in a Diet-Induced Obese Murine Model

    PubMed Central

    Boudoures, Anna L.; Chi, Maggie; Thompson, Alysha; Zhang, Wendy; Moley, Kelle H.

    2016-01-01

    Obesity negatively affects many aspects of the human body, including reproductive function. In females, the root of the decline in fertility is linked to problems in the oocyte. Problems seen in oocytes that positively correlate with increasing BMI include changes to the metabolism, lipid accumulation, meiosis, and metaphase II (MII) spindle structure. Studies in mice indicate dietary interventions fail to reverse these problems [4]. How exercise affects the oocytes has not been addressed. Therefore, we hypothesized an exercise intervention would improve oocyte quality. Here we show in a mouse model of an exercise intervention can improve lipid metabolism in germinal vesicle (GV) stage oocytes. Oocytes significantly increased activity and transcription of the β-oxidation enzyme Hadha (Hydroxyacyl-CoA-dehydrogenase) in response to exercise training only if the mice had been fed a high fat diet (HFD). An exercise intervention also reversed the lipid accumulation seen in GV stage oocytes of HFD females. However, delays in meiosis and disorganized MII spindles remained present. Therefore, exercise is able to improve, but not reverse, damage imparted on oocytes as a result of a high fat diet and obesity. By utilizing an exercise intervention on a HFD, we determined only lipid content and lipid metabolism is changed in GV oocytes. Moving forward, interventions to improve oocyte quality may need to be more targeted to the oocyte specifically. Because of the HFD induced deficiency in β-oxidation, dietary supplementation with substrates to improve lipid utilization may be more beneficial. PMID:26700938

  13. Aging of recipient oocytes reduces the development of cloned embryos receiving cumulus cells.

    PubMed

    Liu, Guohui; Kato, Yoko; Tsunoda, Yukio

    2007-08-01

    Parthenogenetic activation is an important factor in successful production of cloned mammals. Because it has been reported that aged oocytes are more sensitive to parthenogenetic activation than young oocytes, the present study examined the effects of oocyte aging on the in vitro and in vivo developmental potential of nuclear-transferred (NT) mouse oocytes receiving cumulus cells. The potentials of young NT oocytes (14 h after human chorionic gonadotrophin [hCG] injection) to develop into blastocysts was, however, significantly higher than that of aged oocytes (20 h after hCG injection; 16% vs 6%). When the nuclei of NT oocytes at the 2-cell stage were fused with enucleated fertilized 2-cell embryos, the potentials of the serial NT embryos to develop into blastocysts were no different for both young and aged oocytes (74% vs 74%). Live young, however, were obtained only after transfer of serial NT blastocysts developed from young NT oocytes (2%). In contrast to a report using embryonic nuclei as the nuclear donors, the results of the present study indicate that young oocytes are superior to aged oocytes as a source of recipient cytoplasm for mouse somatic cell cloning. PMID:17389775

  14. Glucocorticoids impair oocyte developmental potential by triggering apoptosis of ovarian cells via activating the Fas system

    PubMed Central

    Yuan, Hong-Jie; Han, Xiao; He, Nan; Wang, Guo-Liang; Gong, Shuai; Lin, Juan; Gao, Min; Tan, Jing-He

    2016-01-01

    Previous studies indicate that stress damages oocytes with increased secretion of glucorticoids. However, although injection of female mice with cortisol decreased oocyte competence, exposure of mouse oocytes directly to physiological or stress-induced concentrations of glucorticoids did not affect oocyte maturation and embryo development. This study has explored the mechanisms by which glucocorticoids impair oocyte competence. Female mice were injected with cortisol and the effects of cortisol-injection on oocyte competence, ovarian cell apoptosis and Fas/FasL activation were observed. The results showed that cortisol-injection decreased (a) oocyte developmental potential, (b) the E2/P4 ratio in serum and ovaries, and (c) expression of insulin-like growth factor 1, brain-derived neurotrophic factor and glucocorticoid receptor in mural granulosa cells (MGCs), while increasing levels of (a) cortisol in serum and ovaries, (b) apoptosis in MGCs and cumulus cells (CCs), (c) FasL secretion in ovaries and during oocyte maturation in vitro, and (d) Fas in MGCs, CCs and oocytes. The detrimental effects of cortisol-injection on oocyte competence and apoptosis of MGCs and CCs were significantly relieved when the gld (generalized lymphoproliferative disorder) mice harboring FasL mutations were observed. Together, the results suggested that glucocorticoids impair oocyte competence by triggering apoptosis of ovarian cells via activating the Fas system. PMID:27040909

  15. Sequencing, Annotation and Analysis of the Syrian Hamster (Mesocricetus auratus) Transcriptome

    PubMed Central

    Tchitchek, Nicolas; Safronetz, David; Rasmussen, Angela L.; Martens, Craig; Virtaneva, Kimmo; Porcella, Stephen F.; Feldmann, Heinz

    2014-01-01

    Background The Syrian hamster (golden hamster, Mesocricetus auratus) is gaining importance as a new experimental animal model for multiple pathogens, including emerging zoonotic diseases such as Ebola. Nevertheless there are currently no publicly available transcriptome reference sequences or genome for this species. Results A cDNA library derived from mRNA and snRNA isolated and pooled from the brains, lungs, spleens, kidneys, livers, and hearts of three adult female Syrian hamsters was sequenced. Sequence reads were assembled into 62,482 contigs and 111,796 reads remained unassembled (singletons). This combined contig/singleton dataset, designated as the Syrian hamster transcriptome, represents a total of 60,117,204 nucleotides. Our Mesocricetus auratus Syrian hamster transcriptome mapped to 11,648 mouse transcripts representing 9,562 distinct genes, and mapped to a similar number of transcripts and genes in the rat. We identified 214 quasi-complete transcripts based on mouse annotations. Canonical pathways involved in a broad spectrum of fundamental biological processes were significantly represented in the library. The Syrian hamster transcriptome was aligned to the current release of the Chinese hamster ovary (CHO) cell transcriptome and genome to improve the genomic annotation of this species. Finally, our Syrian hamster transcriptome was aligned against 14 other rodents, primate and laurasiatheria species to gain insights about the genetic relatedness and placement of this species. Conclusions This Syrian hamster transcriptome dataset significantly improves our knowledge of the Syrian hamster's transcriptome, especially towards its future use in infectious disease research. Moreover, this library is an important resource for the wider scientific community to help improve genome annotation of the Syrian hamster and other closely related species. Furthermore, these data provide the basis for development of expression microarrays that can be used in functional

  16. Femtosecond laser based enucleation of porcine oocytes for somatic cell nuclear transfer

    NASA Astrophysics Data System (ADS)

    Kütemeyer, K.; Lucas-Hahn, A.; Petersen, B.; Hassel, P.; Lemme, E.; Niemann, H.; Heisterkamp, A.

    2009-07-01

    Cloning of several mammalian species has been achieved by somatic cell nuclear transfer (SCNT) in recent years. However, this method still results in very low efficiencies around 1% which originate from suboptimal culture conditions and highly invasive techniques for oocyte enucleation and injection of the donor cell using micromanipulators. In this paper, we present a new minimal invasive method for oocyte imaging and enucleation based on the application of femtosecond (fs) laser pulses. After imaging of the oocyte with multiphoton microscopy, ultrashort pulses are focused onto the metaphase plate of MII-oocytes in order to ablate the DNA molecules. We show that fs laser based enucleation of porcine oocytes completely inhibits the first mitotic cleavage after parthenogenetic activation while maintaining intact oocyte morphology in most cases. In contrast, control groups without previous irradiation of the metaphase plate are able to develop to the blastocyst stage. Further experiments have to clarify the suitability of fs laser based enucleated oocytes for SCNT.

  17. Role of animal pole protuberance and microtubules during meiosis in sea cucumber Apostichopus japonicus oocytes

    NASA Astrophysics Data System (ADS)

    Pang, Zhenguo; Chang, Yaqing; Sun, Huiling; Yu, Jiaping

    2010-05-01

    Fully grown oocytes of Apostichopus japonicus have a cytoplasmic protuberance where the oocyte attaches to the follicle. The protuberance and the oolamina located on the opposite side of the oocyte indicate the animal-vegetal axis. Two pre-meiotic centrosomes are anchored to the protuberance by microtubules between centrosomes and protuberance. After meiosis reinitiation induced by DTT solution, the germinal vesicle (GV) migrates towards the protuberance. The GV breaks down after it migrates to the oocyte membrane on the protuberance side. The protuberance then contracts back into the oocyte and the first polar body extrudes from the site of the former protuberance. The second polar body forms beneath the first. Thus the oocyte protuberance indicates the presumptive animal pole well before maturation of the oocyte.

  18. Mitochondria Synthesize Melatonin to Ameliorate Its Function and Improve Mice Oocyte's Quality under in Vitro Conditions.

    PubMed

    He, Changjiu; Wang, Jing; Zhang, Zhenzhen; Yang, Minghui; Li, Yu; Tian, Xiuzhi; Ma, Teng; Tao, Jingli; Zhu, Kuanfeng; Song, Yukun; Ji, Pengyun; Liu, Guoshi

    2016-01-01

    The physiology of oocyte in vitro maturation remains elusive. Generally, the oocytes have a very low maturation rate under in vitro conditions. In the current study, we found that melatonin promotes the maturation of oocytes in which mitochondria play a pivotal role. It was identified that; (1) mitochondria are the major sites for melatonin synthesis in oocytes and they synthesize large amounts of melatonin during their maturation; (2) melatonin improves mitochondrial function by increased mtDNA copy, mitochondrial membrane potential (ΔΨm) and mitochondrial distribution and ATP production in oocytes; (3) the meiotic spindle assembly is enhanced; (4) melatonin reduces ROS production and inhibits 8-oxodG formation, thereby protecting potential DNA mutation from oxidative damage. As a result, melatonin improves the quality of oocytes, significantly accelerates the developmental ability of IVF embryo. The results provide novel knowledge on the physiology of oocyte's maturation, especially under in vitro conditions. PMID:27314334

  19. Is a decrease in cyclic AMP a necessary and sufficient signal for maturation of amphibian oocytes

    SciTech Connect

    Gelerstein, S.; Shapira, H.; Dascal, N.; Yekuel, R.; Oron, Y.

    1988-05-01

    Acetylcholine rapidly lowered the intracellular levels of cyclic AMP in stage 5 and 6 Xenopus laevis oocytes. Acetylcholine alone did not induce oocyte maturation, though it did accelerate maturation induced by progesterone. The effect of acetylcholine on oocyte maturation was independent of extracellular calcium concentration. Adenosine increased cyclic AMP and abolished the progesterone-induced decrease in cyclic AMP levels in follicles and in denuded oocytes. This effect of adenosine was blocked by the Ra purinergic receptor antagonist, theophylline. Despite those effects, adenosine alone induced maturation in stage 6 oocytes and accelerated progesterone-induced maturation in both stage 5 and 6 cells. Adenosine also induced a significant increase in the rate of /sup 45/Ca efflux from oocytes in the presence and the absence of external calcium. We suggest that the activation of cell surface receptors involved in the release of calcium from cellular stores may induce or accelerate oocyte maturation independently of small changes in intracellular cyclic AMP concentration.

  20. Aurora B regulates spindle bipolarity in meiosis in vertebrate oocytes

    PubMed Central

    Shao, Hua; Ma, Chunqi; Zhang, Xuan; Li, Ruizhen; Miller, Ann L.; Bement, William M.; Liu, X. Johné

    2012-01-01

    Aurora B (Aur-B) plays multiple roles in mitosis, of which the best known are to ensure bi-orientation of sister chromatids by destabilizing incorrectly attached kinetochore microtubules and to participate in cytokinesis. Studies in Xenopus egg extracts, however, have indicated that Aur-B and the chromosome passenger complex play an important role in stabilizing chromosome-associated spindle microtubules. Aur-B stabilizes spindle microtubules in the egg extracts by inhibiting the catastrophe kinesin MCAK. Whether or not Aur-B plays a similar role in intact oocytes remains unknown. Here we have employed a dominant-negative Aur-B mutant (Aur-B122R, in which the ATP-binding lysine122 is replaced with arginine) to investigate the function of Aur-B in spindle assembly in Xenopus oocytes undergoing meiosis. Overexpression of Aur-B122R results in short bipolar spindles or monopolar spindles, with higher concentrations of Aur-B122R producing mostly the latter. Simultaneous inhibition of MCAK translation in oocytes overexpressing Aur-B122R results in suppression of monopolar phenotype, suggesting that Aur-B regulates spindle bipolarity by inhibiting MCAK. Furthermore, recombinant MCAK-4A protein, which lacks all four Aur-B phosphoryaltion sites and is therefore insensitive to Aur-B inhibition but not wild-type MCAK, recapitulated the monopolar phenotype in the oocytes. These results suggest that in vertebrate oocytes that lack centrosomes, one major function of Aur-B is to stabilize chromosome-associated spindle microtubules to ensure spindle bipolarity. PMID:22751439

  1. Detection of condensin I and II in maturing pig oocytes.

    PubMed

    Lisková, Lucie; Susor, Andrej; Pivonková, Katerina; Sasková, Adéla; Karabínová, Pavla; Kubelka, Michal

    2010-01-01

    The multiprotein complexes known as condensins (I and II) are major players in chromosome dynamics in mitotic and meiotic cells. Here, we report for the first time the detection of different condensin subunits from both complexes in mammalian oocytes. Using immunoblotting analysis we examined expression levels of condensin subunits during meiotic maturation of porcine oocytes. The expression of the core subunit structural maintenance of chromosomes 2 (SMC2), identical in both condensin complexes, did not change significantly during maturation. Similarly, there was no significant change in the expression of the chromosome associated protein (CAP)-H and CAP-H2 subunits, components of condensin I and II, respectively. Conversely, the expression profiles of CAP-G, CAP-D2 (condensin I) and CAP-D3 (condensin II) were more interesting. At least two isoforms of the CAP-D2 subunit were detected, along with three isoforms of the CAP-D3 and CAP-G subunits. We suggest that this diverse migration of subunit isoforms is due to post-translational modification. Earlier, it was reported that non-SMC proteins are phosphorylated by cyclin-dependent kinase 1. In the present study, we analysed the phosphorylation status of the three subunits in oocyte extracts using alkaline phosphatase treatment and we found that at least the fastest migrating form of CAP-D3 was likely to be phosphorylated in maturing porcine oocytes. In addition, the localisation of CAP-H and CAP-H2 subunits was examined using immunofluorescence staining with specific antibodies, as well as following microinjection of their enhanced green fluorescent protein-tagged mRNA into germinal vesicle-stage oocytes. CAP-H was found in the cytoplasm, whereas CAP-H2 was localised within the nucleus.

  2. Aurora kinase A controls meiosis I progression in mouse oocytes

    PubMed Central

    Saskova, Adela; Solc, Petr; Baran, Vladimir; Kubelka, Michal; Schultz, Richard M.; Motlik, Jan

    2011-01-01

    Aurora kinase A (AURKA), which is a centrosome-localized serine/threonine kinase crucial for cell cycle control, is critically involved in centrosome maturation and spindle assembly in somatic cells. Active T288 phosphorylated AURKA localizes to the centrosome in the late G2 and also spreads to the minus ends of mitotic spindle microtubules. AURKA activates centrosomal CDC25B and recruits cyclin B1 to centrosomes. We report here functions for AURKA in meiotic maturation of mouse oocytes, which is a model system to study the G2 to M transition. Whereas AURKA is present throughout the entire GV-stage oocyte with a clear accumulation on microtubule organizing centers (MTOC), active AURKA becomes entirely localized to MTOCs shortly before germinal vesicle breakdown. In contrast to somatic cells in which active AURKA is present at the centrosomes and minus ends of microtubules, active AURKA is mainly located on MTOCs at metaphase I (MI) in oocytes. Inhibitor studies using Roscovitine (CDK1 inhibitor), LY-294002 (PI3K inhibitor) and SH-6 (PKB inhibitor) reveal that activation of AURKA localized on MTOCs is independent on PI3K-PKB and CDK1 signaling pathways and MOTC amplification is observed in roscovitine- and SH-6-treated oocytes that fail to undergo nuclear envelope breakdown. Moreover, microinjection of Aurka mRNA into GV-stage oocytes cultured in 3-isobutyl-1-methyl xanthine (IBMX)-containing medium to prevent maturation also results in MOTC amplification in the absence of CDK1 activation. Overexpression of AURKA also leads to formation of an abnormal MI spindle, whereas RNAi-mediated reduction of AURKA interferes with resumption of meiosis and spindle assembly. Results of these experiments indicate that AURKA is a critical MTOC-associated component involved in resumption of meiosis, MTOC multiplication, proper spindle formation and the metaphase I-metaphase II transition. PMID:18677115

  3. Aurora kinase A controls meiosis I progression in mouse oocytes.

    PubMed

    Saskova, Adela; Solc, Petr; Baran, Vladimir; Kubelka, Michal; Schultz, Richard M; Motlik, Jan

    2008-08-01

    Aurora kinase A (AURKA), which is a centrosome-localized serine/threonine kinase crucial for cell cycle control, is critically involved in centrosome maturation and spindle assembly in somatic cells. Active T288 phosphorylated AURKA localizes to the centrosome in the late G(2) and also spreads to the minus ends of mitotic spindle microtubules. AURKA activates centrosomal CDC25B and recruits cyclin B1 to centrosomes. We report here functions for AURKA in meiotic maturation of mouse oocytes, which is a model system to study the G(2) to M transition. Whereas AURKA is present throughout the entire GV-stage oocyte with a clear accumulation on microtubule organizing centers (MTOC), active AURKA becomes entirely localized to MTOCs shortly before germinal vesicle breakdown. In contrast to somatic cells in which active AURKA is present at the centrosomes and minus ends of microtubules, active AURKA is mainly located on MTOCs at metaphase I (MI) in oocytes. Inhibitor studies using Roscovitine (CDK1 inhibitor), LY-294002 (PI3K inhibitor) and SH-6 (PKB inhibitor) reveal that activation of AURKA localized on MTOCs is independent on PI3K-PKB and CDK1 signaling pathways and MOTC amplification is observed in roscovitine- and SH-6-treated oocytes that fail to undergo nuclear envelope breakdown. Moreover, microinjection of Aurka mRNA into GV-stage oocytes cultured in 3-isobutyl-1-methyl xanthine (IBMX)-containing medium to prevent maturation also results in MOTC amplification in the absence of CDK1 activation. Overexpression of AURKA also leads to formation of an abnormal MI spindle, whereas RNAi-mediated reduction of AURKA interferes with resumption of meiosis and spindle assembly. Results of these experiments indicate that AURKA is a critical MTOC-associated component involved in resumption of meiosis, MTOC multiplication, proper spindle formation and the metaphase I-metaphase II transition.

  4. Cytomegalovirus keratitis after penetrating keratoplasty.

    PubMed

    Wehrly, S R; Manning, F J; Proia, A D; Burchette, J L; Foulks, G N

    1995-11-01

    We report the development of cytomegalovirus (CMV) keratitis in the penetrating keratoplasty of a 59-year-old human immunodeficiency virus-negative woman after uncomplicated corneal transplantation. Immunosuppression with topical cyclosporine A 2% in corn oil and topical prednisolone acetate 1% suspension was used postoperatively. The 15-month postoperative course was complicated by multiple episodes of endothelial rejection, medically controlled elevated intraocular pressure, polymicrobial bacterial (coagulase-negative staphlococcus and alpha-hemolytic streptococcus) keratitis, and endothelial plaque formation with associated hypopyon and epithelial defect. The graft failed and penetrating keratoplasty was repeated. Cytomegalovirus infection of superficial keratocytes in a region of scarring was identified in histological sections stained with hematoxylin and eosin and confirmed using mouse monoclonal anti-cytomegalovirus antibodies. Excision of the diseased corneal button with no additional treatment appears to have been curative. Low-grade keratitis was the only manifestation of the CMV infection, and it has not recurred 6 months postoperatively.

  5. Weld penetration and defect control

    SciTech Connect

    Chin, B.A.

    1992-05-15

    Highly engineered designs increasingly require the use of improved materials and sophisticated manufacturing techniques. To obtain optimal performance from these engineered products, improved weld properties and joint reliability are a necessarily. This requirement for improved weld performance and reliability has led to the development of high-performance welding systems in which pre-programmed parameters are specified before any welding takes place. These automated systems however lack the ability to compensate for perturbations which arise during the welding process. Hence the need for systems which monitor and control the in-process status of the welding process. This report discusses work carried out on weld penetration indicators and the feasibility of using these indicators for on-line penetration control.

  6. [Penetrating injuries to the pelvis].

    PubMed

    Doll, D; Lenz, S; Exadaktylos, A K; Stettbacher, A; Degiannis, E; Düsel, W; Siewert, J R

    2006-09-01

    As criminality and weapon use increase, general and military surgeons are increasingly confronted with penetrating pelvic injuries both at home and on peacekeeping missions. Penetrating injuries to the iliac vascular axis are associated with considerable mortality, and thus the majority of these emergency patients arrive in a state of deep hypovolemic shock. Concomitant bowel injuries are present in one of five cases, resulting in contamination of the damaged area. Surgical options are simple lateral repair, ligation of the veins, temporary shunt insertion, and prosthetic graft interposition in the injured artery. In extremis ligation of the common or external iliac artery may be the only option to save the patient's life. Surgeons must be aware that damage control surgery and related methods may be needed early on to enable patient survival. PMID:16906417

  7. Long-term radioimmunotherapy studies of Cu-64 anti-colon carcinoma monoclonal antibody (MAb)-1A3, intact and F(ab{prime}){sub 2} singly and in combination, in the GW39-hamster model

    SciTech Connect

    Connett, J.M.; Anderson, C.J.; Guo, L.W.

    1996-05-01

    In previous studies we have shown that Cu-64 has potential for use in radioimmunotherapy (RIT). The present study was undertaken to examine the therapeutic potential of Cu-64-benzyl-TETA-MAb 1A3, intact and F(ab{prime}){sub 2} fragments, injected single or in combination. Using the model of hamsters carrying the GW39 human colon carcinoma in their thighs, we were interested in whether injecting Cu-64-MAb 1A3 intact and F(ab{prime}){sub 2} fragments together would give improved RIT results compared to either agent alone due to the better tumor penetrating properties of F(ab{prime}){sub 2} fragments and the higher uptake and long tumor residence time of intact MAbs. Hamsters were injected with either 1.5 mCi Cu-64-1A3, 1.5 mCi Cu-64-1A3 F(ab{prime}){sub 2} or a combination of 0.75 mCi Cu-64-1A3 intact and 0.75 mCi Cu-64-1A3 F(ab{prime}){sub 2}. These suboptimal doses of Cu-64 were administered in order to detect any enhanced RIT effects with the combination of Cu-64-labeled MAb and fragments. Control groups received saline along. Hamsters were sacrificed when tumors were > 10 g or after surviving for 6 months. Mean lifespans for hamsters treated with Cu-64-1A3 intact, F(ab{prime}){sub 2}, and the combination were 92 {plus_minus} 44 days, 104 {plus_minus} 54 days and 129 {plus_minus} 48 days respectively, compared to 32 {plus_minus} 5 days for the saline controls (p,0.001). 6 months following treatment 43% of the hamsters (3/7) treated with 1.5 mCi Cu-64 1A3 F(ab{prime}){sub 2}, and 50% of hamsters (5/10) treated with 0.75 mCi Cu-64-1A3 and 0.75 mCi Cu-64-1A3 F(ab{prime}){sub 2} in combination were alive and tumor free. Although tumor grown inhibition was also seen in the group receiving 1.5 mCi Cu-64 1A3 intact, only one hamster (1/7) survived tumor free to 6 months. Results show that Cu-64-1A3 F(ab{prime}){sub 2} as well as intact Cu-64-1A3 can increase survival and effect long term tumor inhibition.

  8. Jeeps Penetrating a Hostile Desert

    ERIC Educational Resources Information Center

    Bailey, Herb

    2009-01-01

    Several jeeps are poised at base camp on the edge of a desert aiming to escort one of them as far as possible into the desert, while the others return to camp. They all have full tanks of gas and share their fuel to maximize penetration. In a friendly desert it is best to leave caches of fuel along the way to help returning jeeps. We solve the…

  9. An ideal oocyte activation protocol and embryo culture conditions for somatic cell nuclear transfer using sheep oocytes.

    PubMed

    Patel, Hiren; Chougule, Shruti; Chohan, Parul; Shah, Naval; Bhartiya, Deepa

    2014-10-01

    Pluripotent stem cells are possibly the best candidates for regenerative medicine, and somatic cell nuclear transfer (SCNT) is one of the viable options to make patient-specific embryonic stem cells. Till date efficacy of SCNT embryos is very low and requires further improvement like ideal oocyte activation and in vitro culture system. The aim of the present study was to evaluate ideal oocyte activation using different stimulation protocols and to study the effect of cumulus co-culture conditions on embryo development. Results demonstrate that between electric stimulation and chemical stimulation using calcium ionomycin and ionophore, best oocyte activation was obtained using calcium ionomycin (5 microM for 5 min) which resulted in 83% cleavage followed by 7% of early blastocyst which further increased to 15% when a cumulus bed was also introduced during embryo culture. Sequential modified Charles Rosenkrans 2 (mCR2) medium was used for embryo culture in which glucose levels were increased from 1 mM to 5 mM from Day 3 onwards. SCNT using cumulus cells as donor somatic cell, calcium ionomycin to activate the reconstructed oocyte and embryo culture on a cumulus bed in sequential mCR2 medium, resulted in the development of 6% embryos to early blastocyst stage. Such technological advances will make SCNT a viable option to make patient-specific pluripotent stem cell lines in near future.

  10. Winter hibernation and UCHL1-p34cdc2 association in toad oocyte maturation competence.

    PubMed

    Kuang, Zhichao; Yao, Yuwei; Shi, Yan; Gu, Zheng; Sun, Zhaogui; Tso, Jiake

    2013-01-01

    Currently, it is believed that toad oocyte maturation is dependent on the physiological conditions of winter hibernation. Previous antibody-blocking experiments have demonstrated that toad ubiquitin carboxyl-terminal hydrolase L1 (tUCHL1) is necessary for germinal vesicle breakdown during toad oocyte maturation. In this paper, we first supply evidence that tUCHL1 is highly evolutionarily conserved. Then, we exclude protein availability and ubiquitin carboxyl-terminal hydrolase enzyme activity as factors in the response of oocytes to winter hibernation. In the context of MPF (maturation promoting factor) controlling oocyte maturation and to further understand the role of UCHL1 in oocyte maturation, we performed adsorption and co-immunoprecipitation experiments using toad oocyte protein extracts and determined that tUCHL1 is associated with MPF in toad oocytes. Recombinant tUCHL1 absorbed p34(cdc2), a component of MPF, in obviously larger quantities from mature oocytes than from immature oocytes, and p13(suc1) was isolated from tUCHL1 with a dependence on the ATP regeneration system, suggesting that still other functions may be involved in their association that require phosphorylation. In oocytes from hibernation-interrupted toads, the p34(cdc2) protein level was significantly lower than in oocytes from toads in artificial hibernation, providing an explanation for the different quantities isolated by recombinant tUCHL1 pull-down and, more importantly, identifying a mechanism involved in the toad oocyte's dependence on a low environmental temperature during winter hibernation. Therefore, in toads, tUCHL1 binds p34(cdc2) and plays a role in oocyte maturation. However, neither tUCHL1 nor cyclin B1 respond to low temperatures to facilitate oocyte maturation competence during winter hibernation.

  11. Efficacy of oocyte vitrification combined with blastocyst stage transfer in an egg donation program.

    PubMed

    García, Javier I; Noriega-Portella, Luis; Noriega-Hoces, Luis

    2011-04-01

    BACKGROUND A successful oocyte vitrification program is important for women with various indications for assisted reproduction technology. The objective of this study was to report the outcome of vitrification of oocytes, obtained through an oocyte donation program, by evaluating the embryo development, pregnancy and implantation rates (IRs) after blastocyst transfer. METHODS A total of 1098 oocytes were obtained from 78 donors. There were 312 oocytes used in the study group (vitrified oocytes) and 786 used in the control group (fresh oocytes). There were 34 recipients who received blastocysts obtained from vitrified oocytes and 58 recipients who received blastocysts from fresh oocytes. The fertilization rate, cleavage rate, embryo quality, pregnancy rate (PR) and IR were compared between groups. RESULTS Vitrified oocytes showed a survival rate of 89.4%. There was no difference in the fertilization rate (76.1 and 87.5%), Day 2 cleavage rate (96.3 and 98.0%) or blastocyst formation rate (41.3 and 45.3%) for the study and control groups, respectively. PRs, IRs and miscarriages rates (MRs) were similar for the study group compared with the control group (PR: 61.8 versus 60.0%; IR: 43.9 versus 42.9%; MR: 9.5 versus 5.9%). CONCLUSIONS The developmental competence of embryos obtained from vitrified oocytes is not affected by the vitrification procedure, since they preserve the potential to be fertilized and to develop into high-quality blastocysts, similar to embryos from fresh oocytes. The successful clinical outcome indicates the use of this procedure for oocyte donation programs and for oocyte storage in general.

  12. Network Penetration Testing and Research

    NASA Technical Reports Server (NTRS)

    Murphy, Brandon F.

    2013-01-01

    This paper will focus the on research and testing done on penetrating a network for security purposes. This research will provide the IT security office new methods of attacks across and against a company's network as well as introduce them to new platforms and software that can be used to better assist with protecting against such attacks. Throughout this paper testing and research has been done on two different Linux based operating systems, for attacking and compromising a Windows based host computer. Backtrack 5 and BlackBuntu (Linux based penetration testing operating systems) are two different "attacker'' computers that will attempt to plant viruses and or NASA USRP - Internship Final Report exploits on a host Windows 7 operating system, as well as try to retrieve information from the host. On each Linux OS (Backtrack 5 and BlackBuntu) there is penetration testing software which provides the necessary tools to create exploits that can compromise a windows system as well as other operating systems. This paper will focus on two main methods of deploying exploits 1 onto a host computer in order to retrieve information from a compromised system. One method of deployment for an exploit that was tested is known as a "social engineering" exploit. This type of method requires interaction from unsuspecting user. With this user interaction, a deployed exploit may allow a malicious user to gain access to the unsuspecting user's computer as well as the network that such computer is connected to. Due to more advance security setting and antivirus protection and detection, this method is easily identified and defended against. The second method of exploit deployment is the method mainly focused upon within this paper. This method required extensive research on the best way to compromise a security enabled protected network. Once a network has been compromised, then any and all devices connected to such network has the potential to be compromised as well. With a compromised

  13. The effect of harvesting technique on efficiency of oocyte collection and different maturation media on the nuclear maturation of oocytes in camels (Camelus dromedarius).

    PubMed

    Nowshari, Manzoor A

    2005-06-01

    The purpose of this investigation was to develop an efficient method for harvesting oocytes from dromedary camel ovaries and to examine the effect of different maturation media on their subsequent maturation in vitro. Oocytes were collected by aspirating the follicular contents using a needle attached to a syringe (Method I, n=163 ovaries) or to a constant aspirating pressure, applied by a vacuum pump (Method II, n=117 ovaries). Individual follicles were excised from ovaries and follicles were punctured with two needles (Method III, n=117). Oocytes were matured in vitro for 40-42 h. At the end of maturation period, oocytes were denuded of cumulus cells and the proportion of oocytes in metaphase-II (MII) stage was determined. In the second experiment, oocytes collected by the dissection method were matured in Tissue Culture Medium199 (TCM), CR1 or modified Connaught Medical Research Laboratories medium-1066 (CMRL) and their nuclear maturation was evaluated after 40-42 h. The recovery rate of oocytes was higher (P<0.01) with Method III compared with Method I or II (94, 31 and 33%, respectively). A higher proportions of oocytes collected with Method I or II were either completely or partially denuded compared with Method III (31, 14% versus 1%). The proportions of viable oocytes (78, 60 and 70%, respectively) and those showing metaphase II was not different (39, 50 and 46%, respectively, P>0.05) among the three treatment groups. Oocyte maturation rate was higher (P<0.05) when TCM was used compared with CMRL or CR1 medium. There was, however, no difference in the maturation rate for oocytes cultured in CMRL or CR1 medium. It may be concluded that a higher proportion of cumulus enclosed oocytes may be recovered by follicle dissection method compared to aspiration using syringe or pump. The higher recovery rate with a comparable proportion of viable and matured oocytes resulted in the overall increase in the number of matured (MII) oocytes/ovary with follicle dissection

  14. Lack of Negative Effects on Syrian Hamsters and Mongolian Gerbils Housed in the Same Secondary Enclosure

    PubMed Central

    Pritchett-Corning, Kathleen R; Gaskill, Brianna N

    2015-01-01

    In cases where different species might be housed in the same room or secondary enclosure, the Guide for the Care and Use of Laboratory Animals recommends that the animals should be behaviorally compatible and have the same health status. Syrian hamsters and Mongolian gerbils, both desert-dwelling rodents, appear to be reasonable candidates for such a combination. This study was undertaken to evaluate whether housing hamsters and gerbils in the same secondary enclosure is an acceptable practice. Weanling and breeding-age hamsters and gerbils were housed in open-topped cages in an isolator for 5 mo; the isolator also contained with nude and haired mice, which acted as sentinels. Cages housing hamsters and gerbils were rotated between species, and dirty bedding was exchanged between species in an effort to transmit microorganisms. In addition, sentinel mice housed in the isolator were supplied with dirty bedding from both hamsters and gerbils. Neither species showed clinical signs of illness, the health status of neither the hamsters nor the gerbils changed significantly, and the sentinel mice acquired only 2 infectious organisms, a Helicobacter species and Staphylococcus aureus. Both hamsters and gerbils bred successfully when housed together in the same isolator, and no infanticide or mortality was seen. Breeding performance did not differ between isolator breeding and barrier breeding. This study supports the housing of hamsters and gerbils in the same secondary enclosure. PMID:26045450

  15. On the analysis of neonatal hamster tooth germs with the photon microprobe at Daresbury, UK

    NASA Astrophysics Data System (ADS)

    Tros, G. H. J.; Van Langevelde, F.; Vis, R. D.

    1990-04-01

    Complementary to the micro-PIXE experiments performed on hamster tooth germs to elucidate the role of fluoride during the growth, the photon microprobe at Daresbury was used to obtain information on the distribution of Zn. The germs of fluoride-administered hamsters, together with a control group, were analyzed with the micro-synchrotron radiation fluorescence method (micro-SXRF).

  16. Lack of negative effects on Syrian hamsters and Mongolian gerbils housed in the same secondary enclosure.

    PubMed

    Pritchett-Corning, Kathleen R; Gaskill, Brianna N

    2015-05-01

    In cases where different species might be housed in the same room or secondary enclosure, the Guide for the Care and Use of Laboratory Animals recommends that the animals should be behaviorally compatible and have the same health status. Syrian hamsters and Mongolian gerbils, both desert-dwelling rodents, appear to be reasonable candidates for such a combination. This study was undertaken to evaluate whether housing hamsters and gerbils in the same secondary enclosure is an acceptable practice. Weanling and breeding-age hamsters and gerbils were housed in open-topped cages in an isolator for 5 mo; the isolator also contained with nude and haired mice, which acted as sentinels. Cages housing hamsters and gerbils were rotated between species, and dirty bedding was exchanged between species in an effort to transmit microorganisms. In addition, sentinel mice housed in the isolator were supplied with dirty bedding from both hamsters and gerbils. Neither species showed clinical signs of illness, the health status of neither the hamsters nor the gerbils changed significantly, and the sentinel mice acquired only 2 infectious organisms, a Helicobacter species and Staphylococcus aureus. Both hamsters and gerbils bred successfully when housed together in the same isolator, and no infanticide or mortality was seen. Breeding performance did not differ between isolator breeding and barrier breeding. This study supports the housing of hamsters and gerbils in the same secondary enclosure.

  17. Effect of exercise on redistribution and clearance of inhaled particles from hamster lungs

    SciTech Connect

    Sweeney, T.D.; Tryka, A.F.; Brain, J.D. )

    1990-03-01

    Does exercise alter the redistribution and clearance of particles from the lungs Sedentary hamsters and hamsters that were exercise trained by voluntary wheel running for the previous 5 wk were exposed to a 198Au-labeled aerosol for 25 min. Six trained and 6 sedentary animals were killed within 5 min after the exposure (day 0); the same number were killed 5 days later. The trained hamsters ran ad libitum during those 5 days. The lungs of all animals were excised, dried at total lung capacity, sliced into 1-mm-thick sections, and dissected into pieces that were counted for radioactivity and weighed. On day 0, trained hamsters had 80% more particles per milligram of lung than sedentary hamsters, although both were exposed under identical conditions of restraint. After five days, exercising hamsters cleared 38% of the particles present at day 0, whereas sedentary animals removed only 15%. Significant clearance was observed from the middle lung regions of sedentary hamsters and from all lung regions in exercising hamsters. We conclude that exercise can enhance the redistribution and clearance of particles from the lungs; the mechanisms responsible are as yet unclear.

  18. Effects of wheel running on photoperiodic responses of Djungarian hamsters (Phodopus sungorus).

    PubMed

    Scherbarth, Frank; Petri, Ines; Steinlechner, Stephan

    2008-07-01

    Djungarian hamsters (Phodopus sungorus) were exposed to artificial short days either with access to a running wheel (RW) or without. Within 6 weeks RW hamsters considerably increased their body mass, whereas controls showed the typical body mass reduction. Estimation of paired testis weights indicated a decelerated testis regression in RW hamsters. Subsequent locking of RWs for 9 weeks led to a decline in body mass of RW animals in parallel to controls. Daily torpor was almost completely missing in hamsters with initially unlocked wheels. During the final phase when RWs were again unlocked (3 weeks), body mass of exercising hamsters increased again, while controls reached the nadir in body mass. In comparison to equiponderate long-day (LD) controls the relative liver weight of RW hamsters was significantly increased unlike the relative heart weight. However, the latter tended to be higher than in sedentary LD hamsters. A growth-stimulating effect of wheel running was proven by elongated femora in exercising short-day (SD) hamsters compared to SD controls and suggested by exercise-induced elevation of body mass in a further experiment under continuous LD conditions, indicating a growth-promoting effect of wheel running independent from the photoperiod.

  19. The Chemistry of Cold: Mechanisms of Torpor Regulation in the Siberian Hamster.

    PubMed

    Cubuk, Ceyda; Bank, Jonathan H H; Herwig, Annika

    2016-01-01

    Siberian hamsters use spontaneous daily torpor, a state of hypometabolism and hypothermia, to save energy during winter. Multiple neuroendocrine signals set the scene for spontaneous torpor to occur, and several brain areas have been identified as potential sites for torpor regulation. Here, we summarize the known mechanisms of a fascinating physiological state in the Siberian hamster.

  20. Diet affects resting, but not basal metabolic rate of normothermic Siberian hamsters acclimated to winter.

    PubMed

    Gutowski, Jakub P; Wojciechowski, Michał S; Jefimow, Małgorzata

    2011-12-01

    We examined the effect of different dietary supplements on seasonal changes in body mass (m(b)), metabolic rate (MR) and nonshivering thermogenesis (NST) capacity in normothermic Siberian hamsters housed under semi-natural conditions. Once a week standard hamster food was supplemented with either sunflower and flax seeds, rich in polyunsaturated fatty acids (FA), or mealworms, rich in saturated and monounsaturated FA. We found that neither of these dietary supplements affected the hamsters' normal winter decrease in m(b) and fat content nor their basal MR or NST capacity. NST capacity of summer-acclimated hamsters was lower than that of winter-acclimated ones. The composition of total body fat reflected the fat composition of the dietary supplements. Resting MR below the lower critical temperature of the hamsters, and their total serum cholesterol concentration were lower in hamsters fed a diet supplemented with mealworms than in hamsters fed a diet supplemented with seeds. These results indicate that in mealworm-fed hamsters energy expenditure in the cold is lower than in animals eating a seed-supplemented diet, and that the degree of FA unsaturation of diet affects energetics of heterotherms, not only during torpor, but also during normothermy.

  1. Abnormalities of ADP/ATP carrier protein in J-2-N cardiomyopathic hamsters.

    PubMed

    Kato, M; Yang, J; Iwai, T; Tanamura, A; Arino, T; Kawashima, O; Takeda, N

    1993-02-17

    ADP/ATP carrier protein (AAC) is located in the mitochondrial inner membrane and has an important function in mitochondrial energy supply. This protein transports ATP to the cytoplasm and counter transports ADP into the mitochondria. J-2-N cardiomyopathic hamsters were investigated to determine the AAC content in cardiac mitochondria. After recording an electrocardiogram and collecting blood, the cardiac mitochondria were isolated. The mitochondrial membranes were labelled with eosin-5-maleimide (EMA) and separated on SDS polyacrylamide gels. The position of the AAC component was identified by exposing the gel under UV light, and the AAC content was determined by densitometry after staining with Coomassie blue. The AAC content ratio was significantly decreased in both 10-week-old and 1-year survived J-2-N hamsters when compared to control Golden hamster. Among 10-week-old J-2-N hamsters, the decrease in the AAC content ratio was more marked for the animals with more severe myocardial damage. The H(+)-ATPase activities of mitochondrial membrane were higher in 10-week-old J-2-N hamsters than in control hamsters. These results suggest that the decrease of AAC in J-2-N hamster plays an important role in the pathogenesis of cardiomyopathy in J-2-N hamsters. PMID:8455591

  2. Lack of negative effects on Syrian hamsters and Mongolian gerbils housed in the same secondary enclosure.

    PubMed

    Pritchett-Corning, Kathleen R; Gaskill, Brianna N

    2015-05-01

    In cases where different species might be housed in the same room or secondary enclosure, the Guide for the Care and Use of Laboratory Animals recommends that the animals should be behaviorally compatible and have the same health status. Syrian hamsters and Mongolian gerbils, both desert-dwelling rodents, appear to be reasonable candidates for such a combination. This study was undertaken to evaluate whether housing hamsters and gerbils in the same secondary enclosure is an acceptable practice. Weanling and breeding-age hamsters and gerbils were housed in open-topped cages in an isolator for 5 mo; the isolator also contained with nude and haired mice, which acted as sentinels. Cages housing hamsters and gerbils were rotated between species, and dirty bedding was exchanged between species in an effort to transmit microorganisms. In addition, sentinel mice housed in the isolator were supplied with dirty bedding from both hamsters and gerbils. Neither species showed clinical signs of illness, the health status of neither the hamsters nor the gerbils changed significantly, and the sentinel mice acquired only 2 infectious organisms, a Helicobacter species and Staphylococcus aureus. Both hamsters and gerbils bred successfully when housed together in the same isolator, and no infanticide or mortality was seen. Breeding performance did not differ between isolator breeding and barrier breeding. This study supports the housing of hamsters and gerbils in the same secondary enclosure. PMID:26045450

  3. Elimination of microbial flora from conventionally raised Syrian hamsters by antimicrobial agents.

    PubMed

    Srivastava, K K

    1988-04-01

    An attempt was made to identify and eliminate the normal endogenous microflora from conventionally-raised Syrian hamsters. A total of 14 bacterial and 2 yeast species were identified using enrichment, differential and selective media. The elimination of endogenous flora was carried out in two steps. In each step, hamsters were given a filter-sterilized mixture of two or more antibiotics for 10 weeks continuously through drinking water. During the second week, the hamsters were immersed once into a germicidal solution then housed in horizontal laminar-flow units. They were monitored at weekly intervals for the presence or absence of microorganisms in their fecal samples and fur swabs which were cultured on media as mentioned above. In step I, up to four resistant bacteria were cultured. On the basis of the antibiotic sensitivity profile of the surviving bacteria, step II procedure was started. The antibiotic mixture containing Kanamycin and Amphotericin B was fatal for the hamsters in both steps. Within 4 to 5 weeks of step II, all groups of hamsters tested bacteria-free and remained so for the duration of the step. Two weeks following withdrawal of antibiotics from drinking water, only one group of hamsters showed the presence of Escherichia coli. The other groups of hamsters tested bacteria-free for the 5 week duration of the experiment. The autopsies of these hamsters revealed greatly enlarged and thin-walled cecums, a characteristic of the germfree state.

  4. Characterization of the effects of metformin on porcine oocyte meiosis and on AMP-activated protein kinase activation in oocytes and cumulus cells.

    PubMed

    Bilodeau-Goeseels, Sylvie; Magyara, Nora; Collignon, Coralie

    2014-05-01

    The adenosine monophosphate-activated protein kinase (AMPK) activators 5-aminoimidazole-4-carboxamide 1-β-d-ribofuranoside (AICAR) and metformin (MET) inhibit resumption of meiosis in porcine cumulus-enclosed oocytes. The objective of this study was to characterize the inhibitory effect of MET on porcine oocyte meiosis by: (1) determining the effects of an AMPK inhibitor and of inhibitors of signalling pathways involved in MET-induced AMPK activation in other cell types on MET-mediated meiotic arrest in porcine cumulus-enclosed oocytes; (2) determining whether MET and AICAR treatments lead to increased activation of porcine oocyte and/or cumulus cell AMPK as measured by phosphorylation of its substrate acetyl-CoA carboxylase; and (3) determining the effects of inhibition of the AMPK kinase, Ca2+/calmodulin-dependent protein kinase kinase (CaMKK), and Ca2+ chelation on oocyte meiotic maturation and AMPK activation in porcine oocytes and cumulus cells. The AMPK inhibitor compound C (CC; 1 μM) did not reverse the inhibitory effect of AICAR (1 mM) and MET (2 mM) on porcine oocyte meiosis. Additionally, CC had a significant inhibitory effect on its own. eNOS, c-Src and PI-3 kinase pathway inhibitors did not reverse the effect of metformin on porcine oocyte meiosis. The level of acetyl-CoA carboxylase (ACC) phosphorylation in oocytes and cumulus cells did not change in response to culture in the presence of MET, AICAR, CC, the CaMKK inhibitor STO-609 or the Ca2+ chelator BAPTA-AM for 3 h, but STO-609 increased the percentage of porcine cumulus-enclosed oocytes (CEO) that remained at the germinal vesicle (GV) stage after 24 h of culture. These results indicate that the inhibitory effect of MET and AICAR on porcine oocyte meiosis was probably not mediated through activation of AMPK.

  5. A novel phenomenon predicting the entry into a state of hibernation in Syrian hamsters (Mesocricetus auratus).

    PubMed

    Arai, Shigeyuki; Hanaya, Toshiharu; Sakurai, Takeo; Ikeda, Masao; Kurimoto, Masashi

    2005-02-01

    When Syrian hamsters (Mesocricetus auratus) are bred in a cold and short-day environment, most animals go into hibernation after a certain period of time. However, to date it has not been possible to predict which hamster will enter hibernation. In this study, we subcutaneously implanted thermo-loggers in hamsters bred in the cold environment, and recorded the subcutaneous temperature at short intervals until they went into hibernation. A time series analysis of temperature disclosed that a fall of 0.4 to 0.8 degrees C in subcutaneous temperature was seen 5 to 16 days before entering hibernation, and this phenomenon continued for three days or more. No hamster went into the hibernation without displaying this signal. Although the mechanism by which this phenomenon takes place is not clear, it is a sign from the body, which is useful for indicating if a hamster will enter hibernation shortly.

  6. Spontaneous nonneoplastic lesions in control Syrian hamsters in three 24-month long-term carcinogenicity studies.

    PubMed

    McInnes, Elizabeth F; Ernst, Heinrich; Germann, Paul-Georg

    2015-02-01

    Information about the incidence of spontaneously occurring, nonneoplastic background findings in Syrian hamsters is essential if Syrian hamsters are to be used for toxicity studies. Male and female Syrian hamsters of the strain Han:AURA from the Fraunhofer Institute for Toxicology and Experimental Medicine (ITEM) breeding colony were maintained as control animals for carcinogenicity studies and were examined for the presence of nonneoplastic background findings either when they died or when the study was terminated. The nonneoplastic background lesions observed at an incidence of >50% (high), >25% (moderate), and >10% (low) in either male or female animals or in both sexes in one or more long-term studies are detailed. The results are compared to previous published reports of nonneoplastic, spontaneous background lesions in Syrian hamsters. Background information about the incidence of background lesions in Syrian hamsters on short- and long-term studies is useful to both toxicologists and toxicological pathologists.

  7. Phylogenetic conservation of immunoglobulin heavy chains: direct comparison of hamster and mouse Cmu genes.

    PubMed Central

    McGuire, K L; Duncan, W R; Tucker, P W

    1985-01-01

    We have analyzed the JH-Cmu locus of the Syrian hamster by DNA cloning and sequencing. The single Cmu gene is highly homologous to that of the mouse. The hamster equivalents of the JH and switch (S) recombination regions are arranged as in the mouse, but surprisingly are not highly conserved. Also unlike its close murine relative, the Smu regions among inbred hamster strains are not polymorphic. The complete nucleotide sequence of hamster and mouse Cmu genes have been compared to partial Cmu sequences of other species. Conservation within a portion of the 3' untranslated region may signify functional requirements for 3' end processing. Mutational frequencies within exons and introns of hamster and mouse do not support the theory that the rate of DNA transitions to transversions decreases with evolutionary distance. Images PMID:2994005

  8. Real-Time PCR Reveals Rapid Dissemination of Leptospira interrogans after Intraperitoneal and Conjunctival Inoculation of Hamsters.

    PubMed

    Wunder, Elsio A; Figueira, Claudio P; Santos, Gisele R; Lourdault, Kristel; Matthias, Michael A; Vinetz, Joseph M; Ramos, Eduardo; Haake, David A; Picardeau, Mathieu; Dos Reis, Mitermayer G; Ko, Albert I

    2016-07-01

    The pathogen Leptospira interrogans is a highly motile spirochete that causes acute and fulminant infections in humans and other accidental hosts. Hematogenous dissemination is important for infection by the pathogen but remains poorly understood because few animal model studies have used sensitive tools to quantify the bacteria. We evaluated the kinetics of leptospiral infection in Golden Syrian hamsters by a sensitive quantitative real-time PCR (TaqMan) with lipl32 as the target gene. The dissemination and bacterial burden were measured after intraperitoneal infection with a high dose (10(8)) or low dose (2.5 × 10(2)) of leptospires. We also examined the conjunctival challenge route to mimic the natural history of infection. Quantification of leptospires in perfused animals revealed that pathogens were detected in all organs of intraperitoneally infected hamsters, including the eye and brain, within 1 h after inoculation of 10(8) virulent L. interrogans bacteria. Peaks of 10(5) to 10(8) leptospires per gram or per milliliter were achieved in blood and all tissues between day 4 and day 8 after intraperitoneal inoculation of high- and low-dose challenges, respectively, coinciding with macroscopic and histological changes. The conjunctival route resulted in a delay in the time to peak organ burden in comparison to intraperitoneal infection, indicating that although infection could be established, penetration efficiency was low across this epithelial barrier. Surprisingly, infection with a large inoculum of high-passage-number attenuated L. interrogans strains resulted in dissemination to all organs in the first 4 days postinfection, albeit with a lower burden, followed by clearance from the blood and organs 7 days postinfection and survival of all animals. These results demonstrate that leptospiral dissemination and tissue invasion occur. In contrast, development of a critical level of tissue burden and pathology are dependent on the virulence of the infecting

  9. Melatonin attenuates photic disruption of circadian rhythms in Siberian hamsters.

    PubMed

    Ruby, N F; Kang, T; Heller, H C

    1997-10-01

    Body temperature (Tb) was recorded via a biotelemetry system from 28 adult male Siberian hamsters maintained in a light-dark (LD) cycle of 16 h light/day for several months. After Tb was recorded for 3 wk, the LD cycle was phase delayed by extending the light phase by 5 h for 1 day; animals remained on a 16:8 LD cycle for the remainder of the experiment. Hamsters were injected daily with melatonin or vehicle solution for several weeks, beginning either 2 mo after (experiment 1) or on the day of (experiment 2) the phase shift; injections occurred within 30 min of dark onset. In experiment 1, 75% of animals free ran with circadian periods >24 h, beginning on the day of the phase shift, and never reentrained to the LD cycle; no hamsters unambiguously entrained to daily injections. In contrast, 78% of animals in experiment 2 entrained to melatonin injections, and 71% of those animals subsequently reentrained to the photocycle when the injection regimen ended. No vehicle-treated animals entrained to the injection schedule. Melatonin had no effect on daily mean Tb and Tb rhythm amplitude in either experiment; however, melatonin doubled the duration of a hyperthermic response that occurred after each injection. Thus melatonin can prevent loss of entrainment induced by a phase shift of the LD cycle but cannot restore entrainment to free-running animals. Failure to reentrain in the presence of two appropriately coordinated entraining agents also suggests that a phase shift of the photocycle can diminish the sensitivity of the circadian system to both photic and nonphotic input.

  10. Aging, reproduction, and the melatonin rhythm in the Siberian hamster.

    PubMed

    Horton, T H; Yellon, S M

    2001-06-01

    The present study tested the hypothesis that responsiveness to melatonin, the presence of the melatonin rhythm in circulation, and parameters of the GnRH neuron system are sustained across the aging continuum in Siberian hamsters. Afternoon melatonin injections induced testicular atrophy in 42% of aged males compared with 100% of adult males. The proportion of aged males failing to respond to the melatonin injections was similar to the proportion that failed to undergo testicular regression upon exposure to short days. Exposure to short days induced testicular atrophy in juvenile and adult hamsters; however, regression was incomplete or absent in 43% of aged males. The nocturnal rise in melatonin was similar with regard to duration and peak amplitude, and appropriate with respect to photoperiod in 25-day-old juveniles, adult (5 months), and aged (17 months) hamsters. Neither advanced age nor timed melatonin treatments affected GnRH neuron numbers or distribution. Fertility was maintained in aged and adult males to a comparable extent with respect to latency to first litter and number of pups per litter; reproductive success was dramatically reduced in aged compared with adult females. Because melatonin rhythms accurately reflect day length information throughout the continuum from puberty to advanced age, the present evidence suggests that limitations in testis regression in response to short days or exogenous melatonin in a subset of aged males result from a reduced ability to respond to melatonin. In the wild, failure to undergo testicular regression in the presence of shortening day lengths may extend the breeding season of aged males.

  11. Photoperiodic regulation of FGF21 production in the Siberian hamster.

    PubMed

    Samms, Ricardo J; Fowler, Maxine J; Cooper, Scott; Emmerson, Paul; Coskun, Tamer; Adams, Andrew C; Kharitonenkov, Alexei; Tsintzas, Kostas; Ebling, Francis J P

    2014-06-01

    This article is part of a Special Issue "Energy Balance". FGF21 is an endocrine member of the fibroblast growth factor superfamily that has been shown to play an important role in the physiological response to nutrient deprivation. Food restriction enhances hepatic FGF21 production, which serves to engage an integrated response to energy deficit. Specifically, elevated FGF21 levels lead to reduced gluconeogenesis and increased hepatic ketogenesis. However, circulating FGF21 concentrations also paradoxically rise in states of metabolic dysfunction such as obesity. Furthermore, multiple peripheral tissues also produce FGF21 in addition to the liver, raising questions as to its endocrine and paracrine roles in the control of energy metabolism. The objectives of this study were to measure plasma FGF21 concentrations in the Siberian hamster, a rodent which undergoes a seasonal cycle of fattening and body weight gain in the long days (LD) of summer, followed by reduction of appetite and fat catabolism in the short days (SD) of winter. Groups of adult male hamsters were raised in long days, and then exposed to SD for up to 12 weeks. Chronic exposure of LD animals to SD led to a significant increase in circulating FGF21 concentrations. This elevation of circulating FGF21 was preceded by an increase in liver FGF21 protein production evident as early as 4 weeks of exposure to SD. FGF21 protein abundance was also increased significantly in interscapular brown adipose tissue, with a positive correlation between plasma levels of FGF21 and BAT protein abundance throughout the experimental period. Epididymal white adipose tissue and skeletal muscle (gastrocnemius) also produced FGF21, but levels did not change in response to a change in photoperiod. In summary, a natural programmed state of fat catabolism was associated with increased FGF21 production in the liver and BAT, consistent with the view that FGF21 has a role in adapting hamsters to the hypophagic winter state.

  12. Reproductive aging in the Djungarian hamster, Phodopus campbelli.

    PubMed

    Edwards, H E; Tweedie, C J; Terranova, P F; Lisk, R D; Wynne-Edwards, K E

    1998-03-01

    Changes in fertility (delivery success), fecundity (litter size and weaning success), sensitivity to pregnancy-blocking stimuli (mate removal after 24 h), and hypothalamic-pituitary-ovarian function were assessed during reproductive aging in the Djungarian hamster, Phodopus campbelli. All indices of aging occurred earlier in the Djungarian hamster than they do in other laboratory models, including the mouse. Fertility and fecundity were halved by 8 mo of age, and the response to pregnancy-blocking stimuli disappeared by 4-6 mo. Pup birth weight increased with maternal age and decreasing litter size. Older females also tended to lose entire litters rather than to reduce litter size and successfully wean remaining pups. Impaired ovarian function was a major contributor to reproductive aging. Numbers of large antral follicles developing in aged females and in aged females receiving eCG treatment were similar to those in young females. However, a few preovulatory follicles routinely failed to ovulate from each ovary. Healthy preovulatory follicles in old females secreted the large quantities of estradiol-17 beta typical of estrous cycles, but eCG-induced preovulatory follicles did not. As young females of this species have smaller folicular reserves than other rodent species, and follicle numbers in older females are further reduced (with a high incidence of completely degenerate ovaries), follicular exhaustion was coincident with reproductive failure. Neuroendocrine aging of the hypothalamus was also implicated in the loss of a pregnancy-blocking response and the failure of maternal care with increasing maternal age. Similarities to reproductive aging in long-cycle species (including primates) suggest that the Djungarian hamster is a useful laboratory model that deserves further investigation. PMID:9510975

  13. In vitro infection by Ehrlichia ruminantium of baby hamster kidney (BHK), Chinese hamster ovary (CHO-K1) and Madin Darby bovine kidney (MDBK) cells.

    PubMed

    Zweygarth, E; Josemans, A I

    2003-06-01

    The Welgevonden stock of Ehrlichia ruminantium, aetiological agent of heartwater, was propagated in baby hamster kidney (BHK) cells, Chinese hamster ovary (CHO-K1) cells and Madin Darby bovine kidney (MDBK) cells. The cultures required supplementation of the medium with cycloheximide for reliable growth of E. ruminantium. Growth of the Welgevonden stock in BHK and CHO-K1 cells could lead to the development of suspension cultures suitable for the mass production of E. ruminantium for an inactivated elementary body vaccine.

  14. β-hexosaminidase from Xenopus laevis eggs and oocytes: from gene to immunochemical characterization.

    PubMed

    Morales, Enrique S; Krapf, Darío; Botta, Pablo E; Cabada, Marcelo O; Arranz, Silvia E

    2012-12-01

    Glycosidases are present both in sperm and eggs in vertebrates and have been associated with different fertilization steps as gamete binding, egg coat penetration, and polyspermy prevention. In this manuscript, we have analyzed the activity of different glycosidases of Xenopus laevis eggs. The main activity corresponded to N-acetyl-β-D-glucosaminidase (Hex), which was reported to participate both in gamete binding and polyspermy prevention among phylogenetically distant animals. We have raised homologous antibodies against a recombinant N-terminal fragment of a X. laevis Hex, and characterized egg's Hex both by Western blot and immunohistochemical assays. Noteworthy, Hex was mainly localized to the cortex of animal hemisphere of full-grown oocytes and oviposited eggs, and remained unaltered after fertilization. Hex is constituted by different pair arrangements of two subunits (α and β), giving rise to three possible Hex isoforms: A (αβ), B (ββ), and S (αα). However, no information was available regarding molecular identity of Hex in amphibians. We present for the first time the primary sequences of two isoforms of X. laevis Hex. Interestingly, our results suggest that α- and β-like subunits that constitute Hex isoforms could be synthesized from a same gene in Xenopus, by alternative exon use. This finding denotes an evolutionary divergence with mammals, where α and β Hex subunits are synthesized from different genes on different chromosomes. PMID:22753314

  15. Determinants for thermoinducible cell binding and plasmid-encoded cellular penetration detected in the absence of the Yersinia pseudotuberculosis invasin protein.

    PubMed Central

    Isberg, R R

    1989-01-01

    Yersinia pseudotuberculosis inv mutants were analyzed for their ability to bind and penetrate mammalian cell lines. Strains defective for the production of invasin and cured of the Yersinia virulence plasmid pIB1 were extremely defective for entry into the HEp-2 cell line. inv strains harboring the virulence plasmid partially overcame this defect, indicating that the virulence plasmid mediates an invasin-independent pathway for low-level entry into cultured cells. Plasmid-cured inv mutants were able to attach efficiently to mammalian cells after bacterial culture at 37 degrees C but not after culture at a lower temperature. The enhanced cellular binding of inv mutants grown at 37 degrees C did not result in efficient cellular penetration, indicating that invasin-mediated entry is the primary chromosomally encoded pathway responsible for Y. pseudotuberculosis penetration into both HEp-2 and Chinese hamster ovary cells under the assay conditions described here. Images PMID:2543628

  16. Distribution and metabolism of four different dimethylated arsenicals in hamsters

    SciTech Connect

    Naranmandura, Hua; Iwata, Katsuya; Suzuki, Kazuo T.; Ogra, Yasumitsu

    2010-05-15

    Arsenic toxicity and distribution are highly dependent on animal species and its chemical species. Recently, thioarsenical has been recognized in highly toxic arsenic metabolites, which was commonly found in human and animal urine. In the present study, we revealed the mechanism underlying the distribution and metabolism of non-thiolated and thiolated dimethylarsenic compounds such as dimethylarsinic acid (DMA{sup V}), dimethylarsinous acid (DMA{sup III}), dimethylmonothioarsinic acid (DMMTA{sup V}), and dimethyldithioarsinic acid (DMDTA{sup V}) after the administration of them into femoral vein of hamsters. DMA{sup V} and DMDTA{sup V} distributed in organs and body fluids were in their unmodified form, while DMA{sup III} and DMMTA{sup V} were bound to proteins and transformed to DMA{sup V} in organs. On the other hand, DMA{sup V} and DMDTA{sup V} were mostly excreted into urine as their intact form 1 h after post-injection, and more than 70% of the doses were recovered in urine as their intact form. By contrast, less than 8-14% of doses were recovered in urine as DMA{sup V}, while more than 60% of doses were distributed in muscles and target organs (liver, kidney, and lung) of hamsters after the injection of DMMTA{sup V} and DMA{sup III}. However, in red blood cells (RBCs), only a small amount of the arsenicals was distributed (less than 4% of the doses) after the injection of DMA{sup III} and DMMTA{sup V}, suggesting that the DMA{sup III} and DMMTA{sup V} were hardly accumulated in hamster RBCs. Based on these observations, we suggest that although DMMTA{sup V} and DMDTA{sup V} are thioarsenicals, DMMTA{sup V} is taken up efficiently by organs, in a manner different from that of DMDTA{sup V}. In addition, the distribution and metabolism of DMMTA{sup V} are like in manner similar to DMA{sup III} in hamsters, while DMDTA{sup V} is in a manner similar to DMA{sup V}.

  17. Quantitative mutagenesis and mutagen screening with Chinese hamster ovary cells

    SciTech Connect

    Hsie, A.W.; San Sebastian, J.R.; Tan, E.L.

    1980-01-01

    A summary is presented on the development of a specific gene mutation assay, the Chinese hamster ovary cells/hypoxanthine-guanine phosphoribosyl transferase (CHO/HGPRT) system, and the utilization of this system to study structure-activity relationship affecting cytotoxicity and gene mutation by various carcinogens. Then, preliminary development and validation of a Multiplex CHO System for the simultaneous determination of chromosome aberration, sister chromatid exchange in addition to cytotoxicity and gene mutation is presented. The potential use of a CHO/human cell hybrid system for measuring chromosomal deletion and loss is discussed.

  18. Pineal-dependent and -independent effects of photoperiod on immune function in Siberian hamsters (Phodopus sungorus)

    PubMed Central

    Wen, Jarvi C.; Dhabhar, Firdaus S.; Prendergast, Brian J.

    2010-01-01

    Siberian hamsters (Phodopus sungorus) exhibit reproductive and immunological responses to photoperiod. Short (<10-h light/day) days induce gonadal atrophy, increase leukocyte concentrations, and attenuate thermoregulatory and behavioral responses to infection. Whereas hamster reproductive responses to photoperiod are dependent on pineal melatonin secretion, the role of the pineal in short-day induced changes in immune function is not fully understood. To examine this, adult hamsters were pinealectomized (PINx) or sham-PINx, and transferred to short days (9-h light/day; SD) or kept in their natal long-day (15-h light/day; LD) photoperiod. Intact and PINx hamsters housed in LD maintained large testes over the next 12 weeks; sham-PINx hamsters exhibited gonadal regression in SD, and PINx abolished this effect. Among pineal-intact hamsters, blood samples revealed increases in leukocyte, lymphocyte, CD62L+ lymphocyte, and T cell counts in SD relative to LD; PINx did not affect leukocyte numbers in LD hamsters, but abolished the SD increase in these measures. Hamsters were then treated with bacterial lipopolysaccharide (LPS), which induced thermoregulatory (fever), behavioral (anorexia, reductions in nest building), and somatic (weight loss) sickness responses in all groups. Among pineal-intact hamsters, febrile and behavioral responses to LPS were attenuated in SD relative to LD. PINx did not affect sickness responses to LPS in LD hamsters, but abolished the ameliorating effects of SD on behavioral responses to LPS. Surprisingly, PINx failed to abolish the effect of SD on fever. In common with the reproductive system, PINx induces the LD phenotype in most aspects of the immune system. The pineal gland is required for photoperiodic regulation of circulating leukocytes and neural-immune interactions that mediate select aspects of sickness behaviors. PMID:17022983

  19. Lymphocytic choriomeningitis virus infection in fetal, newborn, and young adult Syrian hamsters (Mesocricetus auratus).

    PubMed Central

    Parker, J C; Igel, H J; Reynolds, R K; Lewis, A M; Rowe, W P

    1976-01-01

    The pathogenesis of lymphocytic choriomeningitis virus infection in fetal, newborn, and young adult hamsters was studied. Infected newborn hamsters initially developed a persistent viremia and viruria with titers often in excess of 10(4.0) mean infectious doses/0.03 ml of blood or urine. After week 12 two different patterns of infection became evident. Approximately one-half of the hamsters eventually cleared the infection, whereas the others developed a chronic progressive and ultimalely fatal disease characterized by continuous high-titered viremia and viruria and high titers of virus in their tissues. Complement-fixing antibody and, to a lesser degree, virus-neutralizing antibody coexisted with the viremia. Hamsters with persistently high levels of viremia and viruria developed chronic glomerulonephritis and widespread vasculitis, whereas hamsters that cleared their infections did not develop these lesions. Litters of hamsters born to viremic mothers were invariably infected. Litter sizes were small and breeding effectiveness was reduce; however, vertical, congenital infection was successfully passed through three generations. The course of infection in the congenitally infected hamsters was similar to that in newborn infected hamsters, with all animals producing complement-fixing antibody, some animals being capable of clearing the viremia and remaining healthy, and other animals having persistent viremia and fatal disease. Inoculated young adult hamsters did not become diseased, developed viremia and viruria which persisted up to 3 and 6 months, respectively, and developed complement-fixing antibody by 10 days after infection. The prolonged urinary excretion of large amounts of lymphocytic choriomeningitis virus by asymptomatic, chronically infected hamsters is an important public health consideration when dealing with potential human infection. Images PMID:1270139

  20. Metabolic control of oocyte development: linking maternal nutrition and reproductive outcomes

    PubMed Central

    Liu, Honglin; Gu, Xi; Boots, Christina; Moley, Kelle H.

    2015-01-01

    Obesity, diabetes, and related metabolic disorders are major health issues worldwide. As the epidemic of metabolic disorders continues, the associated medical comorbidities, including the detrimental impact on reproduction, increase as well. Emerging evidence suggests that the effects of maternal nutrition on reproductive outcomes are likely to be mediated, at least in part, by oocyte metabolism. Well-balanced and timed energy metabolism is critical for optimal development of oocytes. To date, much of our understanding of oocyte metabolism comes from the effects of extrinsic nutrients on oocyte maturation. In contrast, intrinsic regulation of oocyte development by metabolic enzymes, intracellular mediators, and transport systems is less characterized. Specifically, decreased acid transport proteins levels, increased glucose/lipid content and elevated reactive oxygen species in oocytes have been implicated in meiotic defects, organelle dysfunction and epigenetic alteration. Therefore, metabolic disturbances in oocytes may contribute to the diminished reproductive potential experienced by women with metabolic disorders. In-depth research is needed to further explore the underlying mechanisms. This review also discusses several approaches for metabolic analysis. Metabolomic profiling of oocytes, the surrounding granulosa cells, and follicular fluid will uncover the metabolic networks regulating oocyte development, potentially leading to the identification of oocyte quality markers and prevention of reproductive disease and poor outcomes in offspring. PMID:25280482

  1. Comparative Proteomic Analysis of Buffalo Oocytes Matured in vitro Using iTRAQ Technique

    PubMed Central

    Chen, Lingsheng; Zhai, Linhui; Qu, Chunfeng; Zhang, Chengpu; Li, Sheng; Wu, Feilin; Qi, Yingzi; Lu, Fenghua; Xu, Ping; Li, Xiangping; Shi, Deshun

    2016-01-01

    To investigate the protein profiling of buffalo oocytes at the germinal vesicle (GV) stage and metaphase II (MII) stage, an iTRAQ-based strategy was applied. A total of 3,763 proteins were identified, which representing the largest buffalo oocytes proteome dataset to date. Among these proteins identified, 173 proteins were differentially expressed in GV oocytes and competent MII oocytes, and 146 proteins were differentially abundant in competent and incompetent matured oocytes. Functional and KEGG pathway analysis revealed that the up-regulated proteins in competent MII oocytes were related to chromosome segregation, microtubule-based process, protein transport, oxidation reduction, ribosome, and oxidative phosphorylation, etc., in comparison with GV and incompetent MII oocytes. This is the first proteomic report on buffalo oocytes from different maturation stages and developmental competent status. These data will provide valuable information for understanding the molecular mechanism underlying buffalo oocyte maturation, and these proteins may potentially act as markers to predict developmental competence of buffalo oocyte during in vitro maturation. PMID:27561356

  2. Optimising vitrification of human oocytes using multiple cryoprotectants and morphological and functional assessment.

    PubMed

    Seet, V Y K; Al-Samerria, S; Wong, J; Stanger, J; Yovich, J L; Almahbobi, G

    2013-01-01

    Oocyte vitrification is a clinical practice that allows preservation of fertility potential in women. Vitrification involves quick cooling using high concentrations of cryoprotectants to minimise freezing injuries. However, high concentrations of cryoprotectants have detrimental effects on oocyte quality and eventually the offspring. In addition, current assessment of oocyte quality after vitrification is commonly based only on the morphological appearance of the oocyte, raising concerns regarding its efficiency. Using both morphological and functional assessments, the present study investigated whether combinations of cryoprotectants at lower individual concentrations result in better cryosurvival rates than single cryoprotectants at higher concentrations. Surplus oocytes from IVF patients were vitrified within 24h after retrieval using the Cryotop method with several cryoprotectants, either individually or in combination. The morphological and functional quality of the vitrified oocytes was investigated using light microscopy and computer-based quantification of mitochondrial integrity, respectively. Oocyte quality was significantly higher using a combination of cryoprotectants than vitrification with individual cryoprotectants. In addition, the quality of vitrified oocyte varied depending on the cryoprotectants and type of combination used. The results of the present study indicate that observations based purely on the morphological appearance of the oocyte to assess the cryosurvival rate are insufficient and sometimes misleading. The outcome will have a significant implication in the area of human oocyte cryopreservation as an important approach for fertility preservation. PMID:22967503

  3. Both diet and gene mutation induced obesity affect oocyte quality in mice

    PubMed Central

    Hou, Yan-Jun; Zhu, Cheng-Cheng; Duan, Xing; Liu, Hong-Lin; Wang, Qiang; Sun, Shao-Chen

    2016-01-01

    Obesity was shown to cause reproductive dysfunctions such as reduced conception, infertility and early pregnancy loss. However, the possible effects of obesity on oocyte quality are still not fully understood. In this study we investigated the effects of both diet and gene mutation induced obesity on impairments in mouse oocyte polarization, oxidative stress, and epigenetic modifications. Our results showed that high-fat diet induced obesity (HFD) and gene mutation induced obesity (ob/ob) could both impair oocyte meiotic maturation, disrupt spindle morphology, and reduce oocyte polarity. Oocytes from obese mice underwent oxidative stress, as shown by high DHE and ROS levels. Abnormal mitochondrial distributions and structures were observed in oocytes from obese groups of mice and early apoptosis signals were detected, which suggesting that oxidative stress had impaired mitochondrial function and resulted in oocyte apoptosis. Our results also showed that 5 mC levels and H3K9 and H3K27 methylation levels were altered in oocytes from obese mice, which indicated that DNA methylation and histone methylation had been affected. Our results showed that both HFD and ob/ob induced obesity affected oocyte maturation and that oxidative stress-induced early apoptosis and altered epigenetic modifications may be the reasons for reduced oocyte quality in obese mice. PMID:26732298

  4. In vitro effects of cilostazol, a phosphodiesterase 3A inhibitor, on mouse oocyte maturation and morphology.

    PubMed

    Taiyeb, Ahmed M; Dees, William L; Ridha-Albarzanchi, Mundhir T; Sayes, Christie M; Kraemer, Duane C

    2014-02-01

    Inhibition of phosphodiesterase 3A (PDE3A) in oocytes has been reported to arrest oocyte maturation and to increase intra-oocyte cyclic adenosine monophosphate levels. Although many PDE3A inhibitors have been found to arrest oocyte maturation in different species, including humans, the most commonly prescribed PDE3A inhibitor named cilostazol (CLZ) has not yet been fully evaluated in reproduction. The present study was designed to investigate the potential inhibitory effects of CLZ on oocyte maturation and morphology in vitro. Antral oocytes were recovered from hyperstimulated mice and allocated to 10 different CLZ concentrations (0.00-67.66 μmol/L). Oocytes were then assessed after 24 and 48 h of incubation for maturation and morphology. Some of the evaluated CLZ concentrations (1.06-4.23 μmol/L) were made similar to those observed in human clinical trials. CLZ arrested oocyte maturation at the germinal vesicle (GV) stage at concentrations as low as 1.06 μmol/L (P < 0.0001). A selective degenerative impact of CLZ targeting arrested oocytes at the GV stage was observed during 24 h of incubation (r = -0.781, P < 0.0001). This was not the case with non-arrested oocytes (r = -0.082, P = 0.64). Such degenerative impact was dose-dependent (P < 0.0001), suggesting a role for cyclic adenosine monophosphate in this degenerative process. The degenerated oocytes were of distorted oolema or fragmented cytoplasm. Based on the experiments, it is concluded that CLZ can inhibit oocyte maturation in vitro, at concentrations similar to those observed in humans taking CLZ, and under such conditions the prolonged maintenance of oocytes at the GV stage is harmful. PMID:24341287

  5. CRL4DCAF1 is required in activated oocytes for follicle maintenance and ovulation.

    PubMed

    Yu, Chao; Xu, Yi-Wen; Sha, Qian-Qian; Fan, Heng-Yu

    2015-02-01

    In mammals, oocytes within the primordial follicles require a number of essential factors to maintain their survival. However, the survival factors for activated oocytes have been poorly characterized. Recently we reported that damaged DNA binding protein-1 (DDB1), the linker subunit of the cullin ring-finger ubiquitin E3 ligase-4 (CRL4) complex, and its substrate adaptor, DDB1-CUL4 associated factor-1 (DCAF1), were essential for primordial follicle maintenance. In this study we specifically deleted these in the oocytes of growing follicles, to investigate if DDB1 and DCAF1 were also survival factors for activated oocytes. In the ovaries of Ddb1(fl/fl);Zp3-Cre mice, the primordial follicle pool was intact, but awakened oocytes and growing follicles beyond the primary stage were rapidly depleted. In the ovaries of Dcaf1(fl/fl);Pten(fl/fl);Gdf9-Cre and Ddb1(fl/fl);Pten(fl/fl);Gdf9-Cre mice, global primordial follicle activation was stimulated by enhanced PI3K signaling, but the awakened oocytes were rapidly lost due to no CRL4(DCAF1) activity. These mouse models provided original evidence that CRL4(DCAF1) was essential for maintaining oocyte survival, not only those in dormancy at the primordial follicle stage, but also naturally awakened oocytes and those awakened by hyper-activation of PI3K signaling. Interestingly, the oocyte-specific Ddb1 or Dcaf1 knockout mice had ovulation defects even before oocyte exhaustion. CRL4(DCAF1) within oocytes was required for cumulus expansion and ovulation-related somatic gene expression in a cell non-autonomous manner. Granulosa cells that surrounded these Ddb1 or Dcaf1-deleted oocytes exhibited increased rates of apoptosis and showed poor responses to ovulation signals. These results suggested that CRL4 in oocytes also regulated granulosa cell functions in a cell non-autonomous manner.

  6. Liver condition of Holstein cows affects mitochondrial function and fertilization ability of oocytes

    PubMed Central

    TANAKA, Hiroshi; TAKEO, Shun; ABE, Takahito; KIN, Airi; SHIRASUNA, Koumei; KUWAYAMA, Takehito; IWATA, Hisataka

    2016-01-01

    The aim of the present study was to examine the fertilization ability and mitochondrial function of oocytes derived from cows with or without liver damage. Oocytes were collected from the ovaries of cows with damaged livers (DL) and those of cows with healthy livers (HL), subjected to in vitro maturation, and fertilized in vitro. A significantly high abnormal fertilization rate was observed for oocytes from DL cows compared to oocytes from HL cows. The time to dissolve the zona pellucida by protease before fertilization was similar between the two liver conditions, whereas after fertilization treatment this time was shorter for DL cows than for HL cows. The percentage of oocytes with equivalent cortical granule distributions underneath the membrane was greater for in vitro matured oocytes from HL cows, whereas an immature distribution pattern was observed for oocytes from DL cows. In addition, a greater percentage of oocytes derived from HL cows released cortical granules following fertilization compared with oocytes from DL cows. Mitochondrial function determined by ATP content and membrane potential were similar at the germinal vesicle stage, but post-in vitro maturation, the oocytes derived from HL cows showed higher values than DL cows. The mitochondrial DNA copy number in oocytes was similar between the two liver conditions for both the germinal vesicle and post-in vitro maturation oocytes. In conclusion, liver damage induces low fertilization, likely because of incomplete cortical granule distribution and release, and the maturation of oocytes from DL cows contain low-functioning mitochondria compared to their HL counterparts. PMID:26832309

  7. Birth of a domestic cat kitten produced by vitrification of lipid polarized in vitro matured oocytes.

    PubMed

    Galiguis, Jason; Gómez, Martha C; Leibo, S P; Pope, C Earle

    2014-06-01

    The ability to cryopreserve oocytes is an effective method to retain valuable genetic material of mammals, including that of endangered animals. Embryos of domestic cats are amenable to cryopreservation, whereas their oocytes are much less cryo-tolerant. The capability of oocytes to survive cryopreservation is affected by several factors, one of which has been hypothesized to be the high concentration of intracellular lipids. To test this hypothesis, in this study we polarized lipids of cat oocytes and tested their cooling and freezing sensitivity. We found that the sensitivity of oocytes to cooling and cryopreservation does appear to be related to their high intracellular lipid content, as indicated by higher cryosurvival and development into blastocysts when intracellular lipids of in vitro matured oocytes were polarized before vitrification. However, polarization of all intracellular lipids was detrimental to development of embryos. Cell numbers in blastocysts derived from fully polarized/vitrified oocytes were significantly lower than those of partially polarized/vitrified or non-vitrified/fresh oocytes. Although embryos derived from fully polarized/vitrified oocytes developed to the blastocyst stage at higher rates than those of partially polarized/vitrified or non-centrifuged/vitrified oocytes, their in vivo developmental competence was compromised. When embryos derived from fully polarized/vitrified oocytes were transferred, although two recipients became pregnant, all implanted embryos were reabsorbed. In contrast, when embryos derived from oocytes that were only partially lipid polarized before vitrification and then were transferred, one recipient did become pregnant and produced a live healthy kitten. The present results suggest that other approaches to altering intra-cellular lipid levels in cat oocytes should be evaluated to improve their functional survival after cryopreservation. PMID:24631204

  8. Effects of cryopreservation on meiotic spindles of oocytes and its dynamics after thawing: clinical implications in oocyte freezing--a review article.

    PubMed

    Chen, S U; Lien, Y R; Chao, K H; Ho, H N; Yang, Y S; Lee, T Y

    2003-04-28

    Embryo freezing has been a successful practice, but oocyte cryopreservation formerly achieved poorer results. This was mainly due to low rates of survival, fertilization, and development. The major dissimilarities for oocytes to embryos are the character of the plasma membrane, the presence of cortical granules, at the metaphase of meiosis II with the spindle system. In addition, the oocytes must be fertilized by sperm at the appropriate time. To improve the survival rate, a refined slow freezing method with increased sucrose concentration would dehydrate oocytes more sufficiently. Vitrification is another approach to prevent ice crystal formation. Intracytoplasmic sperm injection is used to overcome possible zona hardening from the release of cortical granules. The microtubules of meiotic spindles are vulnerable to the thermal changes and would depolymerize. Cryopreserved oocytes exhibited serious disturbances of the microtubules immediately after thawing. Fertilization of oocytes with disorganized spindles could lead to chromosomal aneuploidy, digyny, and arrest of cleavage. After incubation, the microtubules would repolymerize in a time-dependent way. Normal fertilization and development of cryopreserved oocytes improved after appropriate incubation and timing of insemination, compatible with recovery of the spindles. With the improvement of survival, fertilization, and cleavage, oocyte cryopreservation would gain an imperative role.

  9. 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

  10. Expression of Bone Morphogenetic Protein Receptor (BMPR) during Perinatal Ovary Development and Primordial Follicle Formation in the Hamster: Possible Regulation by FSH

    PubMed Central

    Wang, Cheng; Roy, Shyamal K.

    2009-01-01

    To understand whether bone morphogenetic protein plays any role in the formation of primordial follicles in the hamster, we examined the temporal and spatial expression of bone morphogenetic protein receptor (BMPR) mRNA and protein in embryonic (E) 13 through postnatal day (P) 15 ovarian cells and a possible regulation by FSH during the formation of primordial follicles on P8. BMPRIA and BMPRII mRNA levels were significantly higher than that of BMPR1B throughout ovary development. BMPRIA and BMPRII mRNA levels increased significantly on E14 and declined by P5 through P6. Whereas BMPRII mRNA increased again by P7, BMPRIA mRNA levels increased through P8 concurrent with primordial follicle formation. In contrast, BMPRIB mRNA levels increased greater than 10-fold on P7-9, with a further 3-fold increase by P10. BMPR proteins were low in the somatic cells and oocytes on E13 but increased progressively during postnatal development. BMPR expression in somatic cells increased markedly on P8. Whereas BMPRII expression declined by P10 and remained steady thereafter, BMPRIA protein expression fluctuated until P15 when it became low and steady. Overall, BMPRIB immunoreactivity also declined by P10 and then remained low in the interstitial cells through P15. FSH antiserum treatment on E12 significantly attenuated receptor mRNA and protein levels by P8, but equine chorionic gonadotropin replacement on P1 reversed the inhibition. Furthermore, FSH in vitro up-regulated BMPR levels in P4 ovaries. This unique pattern of BMPR expression in the oocytes and somatic cells during perinatal ovary development suggests that BMP may play a regulatory role in primordial follicle formation. Furthermore, FSH may regulate BMP action by modulating the expression of its receptors. PMID:19074578

  11. Management of penetrating brain injury

    PubMed Central

    Kazim, Syed Faraz; Shamim, Muhammad Shahzad; Tahir, Muhammad Zubair; Enam, Syed Ather; Waheed, Shahan

    2011-01-01

    Penetrating brain injury (PBI), though less prevalent than closed head trauma, carries a worse prognosis. The publication of Guidelines for the Management of Penetrating Brain Injury in 2001, attempted to standardize the management of PBI. This paper provides a precise and updated account of the medical and surgical management of these unique injuries which still present a significant challenge to practicing neurosurgeons worldwide. The management algorithms presented in this document are based on Guidelines for the Management of Penetrating Brain Injury and the recommendations are from literature published after 2001. Optimum management of PBI requires adequate comprehension of mechanism and pathophysiology of injury. Based on current evidence, we recommend computed tomography scanning as the neuroradiologic modality of choice for PBI patients. Cerebral angiography is recommended in patients with PBI, where there is a high suspicion of vascular injury. It is still debatable whether craniectomy or craniotomy is the best approach in PBI patients. The recent trend is toward a less aggressive debridement of deep-seated bone and missile fragments and a more aggressive antibiotic prophylaxis in an effort to improve outcomes. Cerebrospinal fluid (CSF) leaks are common in PBI patients and surgical correction is recommended for those which do not close spontaneously or are refractory to CSF diversion through a ventricular or lumbar drain. The risk of post-traumatic epilepsy after PBI is high, and therefore, the use of prophylactic anticonvulsants is recommended. Advanced age, suicide attempts, associated coagulopathy, Glasgow coma scale score of 3 with bilaterally fixed and dilated pupils, and high initial intracranial pressure have been correlated with worse outcomes in PBI patients. PMID:21887033

  12. Demography of penetrating cardiac trauma.

    PubMed Central

    Naughton, M J; Brissie, R M; Bessey, P Q; McEachern, M M; Donald, J M; Laws, H L

    1989-01-01

    All cases of penetrating cardiac trauma in 1985 and 1986 in Jefferson County, Alabama, where patients dying of penetrating trauma received autopsies, were retrospectively reviewed. All hospitals in the county plus the single coroner's office provided the records of the 72 patients comprising this study. Incidents occurred most often in the home or residence (70%) by a known assailant (83%) due to domestic/social disputes (73%). Frequency was greatest in the evening hours (73% between 6:00 PM and 3:00 AM), on weekends in spring and summer. Victims tended to be male (86%), black (72%), married (46%), blue collar workers (62%). There were 41 (57%) gunshot wounds, 3 (4%) shotgun wounds, and 28 (39%) stab wounds with an associated mortality rate of 97%, 100%, and 68%, respectively. Prehospital mortality rate (dead at the scene) was 54.2% (39/72), and death on arrival was 26.4% (19/72), for a combined pretreatment mortality rate of 80.6%. All patients who arrived with no vital signs died. Mortality appeared to be related to mechanism of injury, age, race, sex, vital signs on arrival, number and specific cardiac chambers injured, associated major vascular injury, hematocrit, and mode of transportation. Mortality was not related to caliber of weapon, ethanol level, transport time, time from arrival to operation, or transfusion requirements. There were only ten survivors (1 gunshot wound and 9 stab wounds), all of whom had ventricular injuries and no associated major vascular injuries. The ten survivors represented a 71.4% (10/14) salvage rate for those victims arriving with vital signs. Complications occurred in three patients. Hospitalization averaged 7.3 days in the survivors. Penetrating cardiac trauma remains a serious, socially linked disease with a high rate of mortality. Rapid transport, aggressive resuscitation and cardiorrhaphy remain the best treatment. PMID:2730180

  13. FAA Fluorescent Penetrant Activities - An Update

    SciTech Connect

    Moore, D.G.

    1998-10-20

    The Federal Aviation Administration's Airworthiness Assurance NDI Validation Center (AANC) is currently characterizing low cycle fatigue specimens that will support the needs of penetrant manufacturers, commercial airline industry and the Federal Aviation Administration. The main focus of this characterization is to maintain and enhance the evaluation of penetrant inspection materials and apply resources to support the aircraft community needs. This paper discusses efforts to-date to document the Wright Laboratory penetrant evaluation process and characterize penetrant brightness readings in the initial set of sample calibration panels using Type 1 penetrant.

  14. The efficacy and safety of human oocyte vitrification.

    PubMed

    Nagy, Zsolt Peter; Chang, Ching-Chien; Shapiro, Daniel B; Bernal, Diana Patricia; Kort, Hilton I; Vajta, Gábor

    2009-11-01

    Vitrification is now a widely applied and highly successful approach for cryopreservation in reproductive biology. Rapidly increasing data prove that it is also a highly efficient technique for low-temperature storage of human oocytes. The latest approaches with appropriately selected cryoprotectants, tools and techniques, and properly adjusted parameters allow close to 100% morphological survival rates, and in vitro embryo development, as well pregnancy and implantation rates, comparable with those achieved with fresh oocytes. With standardization of the technique and elimination of biosafety problems by preserving all the positive features, vitrification may become a common part of the everyday routine in a human embryo laboratory, and it may offer a solution for various medical and social situations as well as for simple logistic problems commonly occurring in assisted reproduction. PMID:19806513

  15. Oocyte-specific deletion of Pten in mice reveals a stage-specific function of PTEN/PI3K signaling in oocytes in controlling follicular activation.

    PubMed

    Jagarlamudi, Krishna; Liu, Lian; Adhikari, Deepak; Reddy, Pradeep; Idahl, Annika; Ottander, Ulrika; Lundin, Eva; Liu, Kui

    2009-07-09

    Immature ovarian primordial follicles are essential for maintenance of the reproductive lifespan of female mammals. Recently, it was found that overactivation of the phosphatidylinositol 3-kinase (PI3K) signaling in oocytes of primordial follicles by an oocyte-specific deletion of Pten (phosphatase and tensin homolog deleted on chromosome ten), the gene encoding PI3K negative regulator PTEN, results in premature activation of the entire pool of primordial follicles, indicating that activation of the PI3K pathway in oocytes is important for control of follicular activation. To investigate whether PI3K signaling in oocytes of primary and further developed follicles also plays a role at later stages in follicular development and ovulation, we conditionally deleted the Pten gene from oocytes of primary and further developed follicles by using transgenic mice expressing zona pellucida 3 (Zp3) promoter-mediated Cre recombinase. Our results show that Pten was efficiently deleted from oocytes of primary and further developed follicles, as indicated by the elevated phosphorylation of the major PI3K downstream component Akt. However, follicular development was not altered and oocyte maturation was also normal, which led to normal fertility with unaltered litter size in the mutant mice. Our data indicate that properly controlled PTEN/PI3K-Akt signaling in oocytes is essential for control of the development of primordial follicles whereas overactivation of PI3K signaling in oocytes does not appear to affect the development of growing follicles. This suggests that there is a stage-specific function of PTEN/PI3K signaling in mouse oocytes that controls follicular activation.

  16. Oocyte-specific inactivation of Omcg1 leads to DNA damage and c-Abl/TAp63-dependent oocyte death associated with dramatic remodeling of ovarian somatic cells

    PubMed Central

    Vandormael-Pournin, S; Guigon, C J; Ishaq, M; Coudouel, N; Avé, P; Huerre, M; Magre, S; Cohen-Tannoudji, J; Cohen-Tannoudji, M

    2015-01-01

    Aberrant loss of oocytes following cancer treatments or genetic mutations leads to premature ovarian insufficiency (POI) associated with endocrine-related disorders in 1% of women. Therefore, understanding the mechanisms governing oocyte death is crucial for the preservation of female fertility. Here, we report the striking reproductive features of a novel mouse model of POI obtained through oocyte-specific inactivation (ocKO) of Omcg1/Zfp830 encoding a nuclear zinc finger protein involved in pre-mRNA processing. Genetic ablation of OMCG1 in early growing oocytes leads to reduced transcription, accumulation of DNA double-strand breaks and subsequent c-Abl/TAp63-dependent oocyte death, thus uncovering the key role of OMCG1 for oocyte genomic integrity. All adult Omcg1ocKO females displayed complete elimination of early growing oocytes and sterility. Unexpectedly, mutant females exhibited a normal onset of puberty and sexual receptivity. Detailed studies of Omcg1ocKO ovaries revealed that the ovarian somatic compartment underwent a dramatic structural and functional remodeling. This allowed the cooperation between oocyte-depleted follicles and interstitial tissue to produce estradiol. Moreover, despite early folliculogenesis arrest, mutant mice exhibited sexual cyclicity as shown by cyclical changes in estrogen secretion, vaginal epithelium cytology and genital tract weight. Collectively, our findings demonstrate the key role of Omcg1 for oocyte survival and highlight the contribution of p63 pathway in damaged oocyte elimination in adulthood. Moreover, our findings challenge the prevailing view that sexual cyclicity is tightly dependent upon the pace of folliculogenesis and luteal differentiation. PMID:25168238

  17. Oocyte-somatic cells interactions, lessons from evolution

    PubMed Central

    2012-01-01

    Background Despite the known importance of somatic cells for oocyte developmental competence acquisition, the overall mechanisms underlying the acquisition of full developmental competence are far from being understood, especially in non-mammalian species. The present work aimed at identifying key molecular signals from somatic origin that would be shared by vertebrates. Results Using a parallel transcriptomic analysis in 4 vertebrate species - a teleost fish, an amphibian, and two mammals - at similar key steps of developmental competence acquisition, we identified a large number of species-specific differentially expressed genes and a surprisingly high number of orthologous genes exhibiting similar expression profiles in the 3 tetrapods and in the 4 vertebrates. Among the evolutionary conserved players participating in developmental competence acquisition are genes involved in key processes such as cellular energy metabolism, cell-to-cell communications, and meiosis control. In addition, we report many novel molecular actors from somatic origin that have never been studied in the vertebrate ovary. Interestingly, a significant number of these new players actively participate in Drosophila oogenesis. Conclusions Our study provides a comprehensive overview of evolutionary-conserved mechanisms from somatic origin participating in oocyte developmental competence acquisition in 4 vertebrates. Together our results indicate that despite major differences in ovarian follicular structure, some of the key players from somatic origin involved in oocyte developmental competence acquisition would be shared, not only by vertebrates, but also by metazoans. The conservation of these mechanisms during vertebrate evolution further emphasizes the important contribution of the somatic compartment to oocyte quality and paves the way for future investigations aiming at better understanding what makes a good egg. PMID:23083410

  18. Embryonic development in human oocytes fertilized by split insemination

    PubMed Central

    Kim, Myo Sun; Kim, Jayeon; Youm, Hye Won; Park, Jung Yeon; Choi, Hwa Young

    2015-01-01

    Objective To compare the laboratory outcomes of intracytoplasmic sperm injection (ICSI) and conventional insemination using sibling oocytes in poor prognosis IVF cycles where ICSI is not indicated. Methods Couples undergoing IVF with following conditions were enrolled: history of more than 3 years of unexplained infertility, history of ≥3 failed intrauterine insemination, leukocytospermia or wide variation in semen analysis, poor oocyte quality, or ≥50% of embryos had poor quality in previous IVF cycle(s). Couples with severe male factor requiring ICSI were excluded. Oocytes were randomly assigned to the conventional insemination (conventional group) or ICSI (ICSI group). Fertilization rate (FR), total fertilization failure, and embryonic development at day 3 and day 5 were assessed. Results A total of 309 mature oocytes from 37 IVF cycles (32 couples) were obtained: 161 were assigned to conventional group and 148 to ICSI group. FR was significantly higher in the ICSI group compared to the conventional group (90.5% vs. 72.7%, P<0.001). Total fertilization failure occurred in only one cycle in conventional group. On day 3, the percentage of cleavage stage embryos was higher in ICSI group however the difference was marginally significant (P=0.055). In 11 cycles in which day 5 culture was attempted, the percentage of blastocyst (per cleaved embryo) was significantly higher in the ICSI group than the conventional group (55.9% vs. 25.9%, P=0.029). Conclusion Higher FR and more blastocyst could be achieved by ICSI in specific circumstances. Fertilization method can be tailored accordingly to improve IVF outcomes. PMID:26023671

  19. Calcium and actin in the saga of awakening oocytes

    SciTech Connect

    Santella, Luigia Limatola, Nunzia; Chun, Jong T.

    2015-04-24

    The interaction of the spermatozoon with the egg at fertilization remains one of the most fascinating mysteries of life. Much of our scientific knowledge on fertilization comes from studies on sea urchin and starfish, which provide plenty of gametes. Large and transparent, these eggs have served as excellent model systems for studying egg activation and embryo development in seawater, a plain natural medium. Starfish oocytes allow the study of the cortical, cytoplasmic and nuclear changes during the meiotic maturation process, which can also be triggered in vitro by hormonal stimulation. These morphological and biochemical changes ensure successful fertilization of the eggs at the first metaphase. On the other hand, sea urchin eggs are fertilized after the completion of meiosis, and are particularly suitable for the study of sperm–egg interaction, early events of egg activation, and embryonic development, as a large number of mature eggs can be fertilized synchronously. Starfish and sea urchin eggs undergo abrupt changes in the cytoskeleton and ion fluxes in response to the fertilizing spermatozoon. The plasma membrane and cortex of an egg thus represent “excitable media” that quickly respond to the stimulus with the Ca{sup 2+} swings and structural changes. In this article, we review some of the key findings on the rapid dynamic rearrangements of the actin cytoskeleton in the oocyte/egg cortex upon hormonal or sperm stimulation and their roles in the modulation of the Ca{sup 2+} signals and in the control of monospermic fertilization. - Highlights: • Besides microtubules, microfilaments may anchor the nucleus to oocyte surface. • The cortical Ca{sup 2+} flash and wave at fertilization mirror electrical membrane change. • Artificial egg activation lacks microvilli extension in the perivitelline space. • Calcium is necessary but not sufficient for cortical granules exocytosis. • Actin cytoskeleton modulates Ca{sup 2+} release at oocyte maturation

  20. Purification and characterization of RNase from Rana catesbiana (bullfrog) oocytes

    SciTech Connect

    Liao, Y.D.; Lin, C.T. )

    1991-03-11

    In vitro transcription study under conditions where 5S ribosomal (rRNA) synthesis was highly active in oocyte extracts of X. laevis, the transcriptional activity was not detected in oocyte extracts of cold treated R. catesbiana. The lack of 5S rRNA transcription was not due to the absence of RNA polymerase III, since this enzyme was still active when poly d(A-T) was used as a template. It was found that R. catesbiana extracts could cleave exogenously added 5S rRNA, tRNA and VA-RNA while the X. laevis extract could not. The presence of this RNase activity was not a result of oocyte destruction because eggs derived from R. catesbiana oocytes, which contained this RNase activity, developed into tadpoles after artificial fertilization. In order to elucidate the relationship between the RNase activity and the regulation of gene transcription of cold treated R. catesbiana, this enzyme was purified to homogeneity and characterized. This enzyme could be inactivated by heating to 80C for 15 min, but was resistant to acid and alkaline conditions. The optimal temperature for activity was 55C-65C, while the optimal pH was 4 in 50 mM acetate buffer and 8 in 50 mM Tris-buffer. The optimal cation concentration for the enzyme activity was 4 mM and 0.5 mM for Mg{sup ++} and Zn{sup ++} respectively. The specific cleavage site of this enzyme was located at the phosphodiester bond to the 3{prime} side of the pyrimidine in the pyrimidine-p-G segment. The antiserum against the purified RNase was prepared and used for quantitating this enzyme under different condition.

  1. A hyperpolarization-activated ion current of amphibian oocytes.

    PubMed

    Ochoa-de la Paz, L D; Salazar-Soto, D B; Reyes, J P; Miledi, R; Martinez-Torres, A

    2013-08-01

    A comparative analysis of a hyperpolarization-activated ion current present in amphibian oocytes was performed using the two-electrode voltage-clamp technique in Xenopus laevis, Xenopus tropicalis, and Ambystoma mexicanum. This current appears to be driven mainly by Cl(-) ions, is independent of Ca(2+), and is made evident by applying extremely negative voltage pulses; it shows a slow activating phase and little or no desensitization. The pharmacological profile of the current is complex. The different channel blocker used for Cl(-), K(+), Na(+) and Ca(2+) conductances, exhibited various degrees of inhibition depending of the species. The profiles illustrate the intricacy of the components that give rise to this current. During X. laevis oogenesis, the hyperpolarization-activated current is present at all stages of oocytes tested (II-VI), and the amplitude of the current increases from about 50 nA in stage I to more than 1 μA in stage VI; nevertheless, there was no apparent modification of the kinetics. Our results suggest that the hyperpolarization-activated current is present both in order Anura and Urodela oocytes. However, the electrophysiological and pharmacological characteristics are quite perplexing and seem to suggest a mixture of ionic conductances that includes the activation of both anionic and cationic channels, most probably transiently opened due to the extreme hyperpolarizion of the plasma membrane. As a possible mechanism for the generation of the current, a kinetic model which fits the data suggests the opening of pores in the plasma membrane whose ion selectivity is dependent on the extracellular Cl(-) concentration. The extreme voltage conditions could induce the opening of otherwise latent pores in plasma membrane proteins (i.e., carriers), resembling the ´slippage´ events already described for some carriers. These observations should be valuable for other groups trying to express cloned, voltage-dependent ion channels in oocytes of

  2. The release from metaphase arrest in blue mussel oocytes.

    PubMed

    Néant, I; Dufresne, L; Morasse, J; Gicquaud, C; Guerrier, P; Dubé, F

    1994-09-01

    In Mytilus edulis, shed oocytes are arrested at metaphase I of meiosis until fertilization. We previously suggested (Dubé and Dufresne, J. Exp. Zool. 256:323-332, 1990) that such a metaphase arrest depends upon a continuous synthesis of short-lived proteins, the destruction of which is sufficient to induce meiosis resumption. We further investigated the mechanism of metaphase release in blue mussel oocytes as triggered either by fertilization or by inhibition of protein synthesis (emetine) or phosphorylation (6-dimethylaminopurine, 6-DMAP). Treatment of unfertilized oocytes (UF) with emetine induces completion of the first meiotic cycle including extrusion of the polar body, followed by chromosome decondensation and by the formation of large membrane-bound nuclei, as visualized by Hoechst staining and transmission electron microscopy (TEM). Inhibition of protein phosphorylation with 6-DMAP induces directly chromosome decondensation and the formation of multiple nuclei surrounded by nuclear membrane. These interphasic nuclei exhibit continuous 3H-thymidine incorporation. p13 precipitation of p34 and associated proteins reveals "putative" cyclins in UF, no longer detected after metaphase/anaphase transition due to fertilization or emetine treatment. In the presence of 6-DMAP, new migrating forms are observed. The phosphorylated p34cdc2 homolog becomes dephosphorylated after fertilization or emetine treatment, whereas 6-DMAP induces its phosphorylation on tyrosine. Histone H1 kinase activity is reduced after these treatments, compared to the UF sample. Our results suggest that the metaphase/anaphase transition triggered by fertilization in blue mussel oocytes is induced by the rapid destruction of a set of continuously synthesized proteins accompanied by decreased histone H1 kinase activity. These events can be mimicked by inhibiting protein synthesis. Inhibition of protein phosphorylation would drive the cell to interphase without commitment to meiosis I. PMID

  3. Lipid droplet analysis using in vitro bovine oocytes and embryos.

    PubMed

    Ordoñez-Leon, E A; Merchant, H; Medrano, A; Kjelland, M; Romo, S

    2014-04-01

    The aim of this study was to quantify the content of lipid droplets in bovine oocytes and embryos from Bos indicus (Bi), Bos taurus (Bt) and Bos indicus × Bos taurus (Bi × Bt). Oocytes were aspirated post-mortem and subjected to in vitro maturation, in vitro fertilization and in vitro development; the medium employed at each stage (TCM-199, TALP, SOF) was supplemented with (i) serum replacement (SR), (ii) foetal calf serum (FCS) or (iii) oestrous cow serum (ECS). The structure and distribution of the lipid droplets were established using electron microscopy, but were quantified using an optical microscope on semi-fine toluidine blue-stained sections. The highest percentage of embryos corresponded to those produced with FCS and ECS, which differed from embryos generated with SR (p < 0.05). The highest percentage of morulae and the lowest percentage of blastocysts were obtained with the SR supplement (p < 0.05). The oocytes cultured in FCS demonstrated a higher number of lipid droplets compared to those cultured in SR and ECS (p < 0.05). Less accumulation of lipids was observed in embryos supplemented with SR. The lowest and highest numbers of lipid droplets in oocytes corresponded to the Bi and Bt strain, respectively. The lowest amount of lipid droplets in embryos was observed in Bi (p < 0.05). In conclusion, supplementation of the in vitro development culture medium (synthetic oviduct fluid) with a synthetic substitute serum produced similar results in terms of embryo development compared to those obtained with FCS, but a decreased degree of lipid droplet accumulation was observed in the in vitro-cultured embryos. PMID:24467659

  4. The gametic synapse: RNA transfer to the bovine oocyte.

    PubMed

    Macaulay, Angus D; Gilbert, Isabelle; Caballero, Julieta; Barreto, Rodrigo; Fournier, Eric; Tossou, Prudencio; Sirard, Marc-André; Clarke, Hugh J; Khandjian, Édouard W; Richard, Francois J; Hyttel, Poul; Robert, Claude

    2014-10-01

    Even after several decades of quiescent storage in the ovary, the female germ cell is capable of reinitiating transcription to build the reserves that are essential to support early embryonic development. In the current model of mammalian oogenesis, there exists bilateral communication between the gamete and the surrounding cells that is limited to paracrine signaling and direct transfer of small molecules via gap junctions existing at the end of the somatic cells' projections that are in contact with the oolemma. The purpose of this work was to explore the role of cumulus cell projections as a means of conductance of large molecules, including RNA, to the mammalian oocyte. By studying nascent RNA with confocal and transmission electron microscopy in combination with transcript detection, we show that the somatic cells surrounding the fully grown bovine oocyte contribute to the maternal reserves by actively transferring large cargo, including mRNA and long noncoding RNA. This occurrence was further demonstrated by the reconstruction of cumulus-oocyte complexes with transfected cumulus cells transferring a synthetic transcript. We propose selective transfer of transcripts occurs, the delivery of which is supported by a remarkable synapselike vesicular trafficking connection between the cumulus cells and the gamete. This unexpected exogenous contribution to the maternal stores offers a new perspective on the determinants of female fertility.

  5. Cytoskeletal Dynamics and Fluid Flow in Drosophila Oocytes

    NASA Astrophysics Data System (ADS)

    de Canio, Gabriele; Goldstein, Raymond; Lauga, Eric

    2015-11-01

    The biological world includes a broad range of phenomena in which transport in a fluid plays a central role. Among these is the fundamental issue of cell polarity arising during development, studied historically using the model organism Drosophila melanogaster. The polarity of the oocyte is known to be induced by the translocation of mRNAs by kinesin motor proteins along a dense microtubule cytoskeleton, a process which also induces cytoplasmic streaming. Recent experimental observations have revealed the remarkable fluid-structure interactions that occur as the streaming flows back-react on the microtubules. In this work we use a combination of theory and simulations to address the interplay between the fluid flow and the configuration of cytoskeletal filaments leading to the directed motion inside the oocyte. We show in particular that the mechanical coupling between the fluid motion and the orientation of the microtubules can lead to a transition to coherent motion within the oocyte, as observed. Supported by EPSRC and ERC Advanced Investigator Grant 247333.

  6. Ovarian Germline Stem Cells: An Unlimited Source of Oocytes?

    PubMed Central

    Hanna, Carol; Hennebold, Jon

    2014-01-01

    While there has been progress in directing the development of embryonic stem cells and induced pluripotent stem cells toward a germ cell state, their ability to serve as a source of functional oocytes in a clinically relevant model or situation has yet to be established. Recent studies suggest the adult mammalian ovary is not endowed with a finite number of oocytes, but instead possesses stem cells that contribute to their renewal. The ability to isolate and promote the growth and development of such ovarian germline stem cells (GSCs) would provide a novel means to treat infertility in women. While such ovarian GSCs are well-characterized in non-mammalian model organisms, the findings that support the existence of adult ovarian GSCs in mammals have been met with considerable evidence that disputes their existence. Thus, this review details the lessons provided by model organisms that successfully utilize ovarian GSCs to allow for a continual and high level of female germ cell production throughout their life, with a specific focus on the cellular mechanisms involved in GSC self-renewal and oocyte development. Such an overview of the role oogonial stem cells play in maintaining fertility in non-mammalian species serves as a backdrop for the data generated to-date that supports or disputes the existence of GSCs in mammals as well as the future of this area of research in terms of its potential for any application in reproductive medicine. PMID:24382341

  7. Actin in xenopus oocytes: II. intracellular distribution and polymerizability

    PubMed Central

    Merriam, RW; Clark, TG

    1978-01-01

    The largest oocytes of Xenopus Laevis were broken open in the absence of shearing forces which might transfer actin from particulate to supernatant fractions. Particulate and postmitochondrial supernatant fractions were prepared by centrifugation. SDS-electrophoretic fractionation on polyacrylamide gels and quantitative scanning techniques were used to separate actin and to assay its amount in cellular fractions. The actin has been identified in electrophoretograms by its molecular weight and its binding to DNase I. oocytes contain 1.4-1.7 {um}g of actin per cell, of which up to 88 percent is recovered in the postmitochondrial supernate under a variety of conditions. In the soluble fraction, it represents about 8.8 percent of the total protein. Its concentration in native cytoplasm was directly assayed at 4.1 mg/ml. There is no detectable actin that can be transferred from the particulate to the soluble phase by neutral detergents or ionic conditions that would depolymerize muscle actin. Centrifugation of the soluble oocyte fractions showed that 75-95 percent of the actin can not be sedimented under forces that would pellet filamentous actin. Addition of potassium and magnesium to the cytoplasm, to concentrations that would polymerize muscle actin, does not increase the amount of sedimentable actin. Roughly one-third of the soluble actin is recovered from Sephadex columns at about the position of monomer. About two- thirds is in complexes of 100,000 daltons or greater. PMID:565782

  8. Targeting oocyte maturation to improve fertility in older women.

    PubMed

    Liu, X Johné

    2016-01-01

    Reproductive aging is an increasingly pressing problem facing women in modern society, due to delay in child bearing. According to Statistics Canada, 52% of all Canadian births in 2011 were by women aged 30 years and older, up from 24% in 1981 ( http://www.statcan.gc.ca/pub/91-209-x/2013001/article/11784-eng.htm ). Women older than 35 years of age experience significantly increased risks of infertility, miscarriage and congenital birth defects, mostly due to poor quality of the eggs. Increasingly sophisticated, and often invasive, assisted reproductive technologies (ARTs) have helped millions of women to achieve reproductive success. However, by and large, ARTs do not address the fundamental issue of reproductive aging in women: age-related decline in egg quality. More importantly, ARTs are not, and will never be, the main solution for the general population. Here, I attempt to review the scientific literature on age-related egg quality decline, based mostly on studies in mice and in humans. Emphasis is given to the brief period of time called oocyte maturation, which occurs just prior to ovulation. The rationale for this emphasis is that oocyte maturation represents a critical window where unfavorable ovarian conditions in older females contribute significantly to the decline of egg quality, and that science-based intervention during oocyte maturation represents the best chance of improving egg quality in older women. Finally, I summarize our own work in recent years on peri-ovulatory putrescine supplementation as a possible remedy for reproductive aging.

  9. Distribution and localization of galectin purified from Rana catesbeiana oocytes.

    PubMed

    Uchiyama, H; Komazaki, S; Oyama, M; Matsui, T; Ozeki, Y

    1997-12-01

    Galectins are a family of lectins that recognize beta-D-galactosides independently of calcium ions, and are widely distributed in animals. To characterize a galectin previously purified from oocytes of Rana catesbeiana (American bullfrog), we studied its distribution and localization in several tissues from this frog. Hemagglutination assay and western blotting showed that this lectin is present in many tissues including the liver, skin, kidney, skeletal muscle, and sciatic nerve, but is particularly concentrated in the ovary. Light microscopic immunohistochemistry showed that this lectin is localized in such places as cell-cell junctions, basement membranes, extracellular matrix, or secretory substances in several organs, indicating that this galectin is mainly distributed extracellularly. However, in the ovary, light microscopy showed that this lectin is present in or associated with the yolk platelet. Electron microscopy further revealed that it is localized in the periphery of the yolk platelet (the yolk plasm), but not in the cortical granule. These results indicate that Rana oocytes contain abundant galectin in their yolk platelets in contrast to Xenopus laevis oocytes, which have been found not to contain galectins but other classes of lectins in their yolk platelets and cortical granules.

  10. Axin-1 Regulates Meiotic Spindle Organization in Mouse Oocytes

    PubMed Central

    Liu, Rui; Liu, Yu; Zhang, Fei; Zhang, Zhen; Shen, Yu-Ting; Xu, Lin; Chen, Ming-Huang; Wang, Ya-Long; Xu, Bai-Hui; Yang, Xiang-Jun; Wang, Hai-Long

    2016-01-01

    Axin-1, a negative regulator of Wnt signaling, is a versatile scaffold protein involved in centrosome separation and spindle assembly in mitosis, but its function in mammalian oogenesis remains unknown. Here we examined the localization and function of Axin-1 during meiotic maturation in mouse oocytes. Immunofluorescence analysis showed that Axin-1 was localized around the spindle. Knockdown of the Axin1 gene by microinjection of specific short interfering (si)RNA into the oocyte cytoplasm resulted in severely defective spindles, misaligned chromosomes, failure of first polar body (PB1) extrusion, and impaired pronuclear formation. However, supplementing the culture medium with the Wnt pathway activator LiCl improved spindle morphology and pronuclear formation. Downregulation of Axin1 gene expression also impaired the spindle pole localization of γ-tubulin/Nek9 and resulted in retention of the spindle assembly checkpoint protein BubR1 at kinetochores after 8.5 h of culture. Our results suggest that Axin-1 is critical for spindle organization and cell cycle progression during meiotic maturation in mouse oocytes. PMID:27284927

  11. Ca(2+)(cyt) negatively regulates the initiation of oocyte maturation.

    PubMed

    Sun, Lu; Machaca, Khaled

    2004-04-01

    Ca(2+) is a ubiquitous intracellular messenger that is important for cell cycle progression. Genetic and biochemical evidence support a role for Ca(2+) in mitosis. In contrast, there has been a long-standing debate as to whether Ca(2+) signals are required for oocyte meiosis. Here, we show that cytoplasmic Ca(2+) (Ca(2+)(cyt)) plays a dual role during Xenopus oocyte maturation. Ca(2+) signals are dispensable for meiosis entry (germinal vesicle breakdown and chromosome condensation), but are required for the completion of meiosis I. Interestingly, in the absence of Ca(2+)(cyt) signals oocytes enter meiosis more rapidly due to faster activation of the MAPK-maturation promoting factor (MPF) kinase cascade. This Ca(2+)-dependent negative regulation of the cell cycle machinery (MAPK-MPF cascade) is due to Ca(2+)(cyt) acting downstream of protein kinase A but upstream of Mos (a MAPK kinase kinase). Therefore, high Ca(2+)(cyt) delays meiosis entry by negatively regulating the initiation of the MAPK-MPF cascade. These results show that Ca(2+) modulates both the cell cycle machinery and nuclear maturation during meiosis.

  12. Development of A Questionnaire to Measure Attitude toward Oocyte Donation

    PubMed Central

    Omani Samani, Reza; Mounesan, Leila; Ezabadi, Zahra; Vesali, Samira

    2015-01-01

    Background To our knowledge, there is no valid and comprehensive questionnaire that considers attitude toward oocyte donation (OD). Therefore this study has aimed to design and develop a tool entitled attitude toward donation-oocyte (ATOD-O) to measure attitude toward OD. Materials and Methods This methodological, qualitative research was undertaken on 15 infertile cases. In addition, we performed a literature review and search of various databases. Validity of this questionnaire was conducted by knowledgeable experts who determined indices such as relevancy, clarity, and comprehensiveness. Reliability of the questionnaire was assessed based on the opinions of experts and infertile couples referred to Royan Institute. Results ATOD-O was designed in 52 statements that covered various issues such as the OD process, donor and recipient characteristics, as well as family, emotional, psychological, legal, religious, and socio-economic dimensions. Results were scored as five points: 1 (strongly disagree), 2 (disagree), 3 (somewhat), 4 (agree), and 5 (strongly agree). The overall relevancy of the questionnaire was 97% and clarity was 96%. Overall comprehensiveness was 100%. Conclusion The findings from this preliminary validation study have indicated that ATOD-O is a valid measure for measuring and assessing attitude toward donated oocytes. This questionnaire can be used in studies regarding different groups of a society. PMID:26644863

  13. Role of luteinizing hormone in luteotropic complex of pregnant hamster

    SciTech Connect

    Tamura, H.; Greenwald, G.S.

    1987-04-01

    Hamsters were hypophysectomized on day 4 of pregnancy and injected subcutaneously on days 4-7 with various combinations of 200 ..mu..g prolactin (Prl), 10 ..mu..g follicle-stimulating hormone (FSH), and 20 ..mu..g luteinizing hormone (LH) in polyvinylpyrrolidone (PVP) to decrease its rate of absorption or in saline. End points for luteal function on day 8 were maintenance of pregnancy, serum progesterone (P/sub 4/), luteal weight, and luteal binding for human chorionic gonadotropin, FSH, and Prl. After hypophysectomy, a drastic decline occurred in all parameters including an 89% decrease in luteal weight. Injection of Prl did not maintain pregnancy nor serum P/sub 4/ but partially maintained luteal weight and human chorionic gonadotropin binding sites per corpus luteum. The minimal luteotropic complex of Prl and FSH was effective in maintaining pregnancy and significantly increased serum P/sub 4/ and Prl and FSH receptors but not to control levels. Thus, the luteotropic activity of LH was only demonstrable when it was injected in a long-acting form; when delivered as a bolus, LH (saline) was luteolytic. P/sub 4/ and estradiol were measured by radioimmunoassay. Radioiodinated gonadotropins were prepared. The percentage of tracer reacting with an excess of receptor were 51% of /sup 125/I-FSH and 45.9% of /sup 125/I-hCG using whole homogenates of hamster ovaries.

  14. Sex differences in Siberian hamster ultradian locomotor rhythms.

    PubMed

    Prendergast, Brian J; Stevenson, Tyler J; Zucker, Irving

    2013-02-17

    Sex differences in ultradian activity rhythms (URs) and circadian rhythms (CRs) were assessed in Siberian hamsters kept in long day (LD) or short day (SD) photoperiods for 40 weeks. For both sexes URs of locomotor activity were more prevalent, greater in amplitude and more robust in SDs. The UR period was longer in females than males in both day lengths. The reproductive system underwent regression and body mass declined during the initial 10 weeks of SD treatment, and in both sexes these traits spontaneously reverted to the LD phenotype at or before 40 weeks in SD, reflecting the development of neuroendocrine refractoriness to SD patterns of melatonin secretion. Hamsters of both sexes, however, continued to display SD-like URs at the 40 weeks time point. CRs were less prevalent and the waveform less robust and lower in amplitude in SDs than LDs; the SD circadian waveform also did not revert to the long-day phenotype after 40 weeks of SD treatment. Short day lengths enhanced ultradian and diminished circadian rhythms in both sexes. Day length controls several UR characteristics via gonadal steroid and melatonin-independent mechanisms. Sex differences in ultradian timing may contribute to sex diphenisms in rhythms of sleep, food intake and exercise.

  15. Social forces can impact the circadian clocks of cohabiting hamsters

    PubMed Central

    Paul, Matthew J.; Indic, Premananda; Schwartz, William J.

    2014-01-01

    A number of field and laboratory studies have shown that the social environment influences daily rhythms in numerous species. However, underlying mechanisms, including the circadian system's role, are not known. Obstacles to this research have been the inability to track and objectively analyse rhythms of individual animals housed together. Here, we employed temperature dataloggers to track individual body temperature rhythms of pairs of cohabiting male Syrian hamsters (Mesocricetus auratus) in constant darkness and applied a continuous wavelet transform to determine the phase of rhythm onset before, during, and after cohabitation. Cohabitation altered the predicted trajectory of rhythm onsets in 34% of individuals, representing 58% of pairs, compared to 12% of hamsters single-housed as ‘virtual pair’ controls. Deviation from the predicted trajectory was by a change in circadian period (τ), which tended to be asymmetric—affecting one individual of the pair in nine of 11 affected pairs—with hints that dominance might play a role. These data implicate a change in the speed of the circadian clock as one mechanism whereby social factors can alter daily rhythms. Miniature dataloggers coupled with wavelet analyses should provide powerful tools for future studies investigating the principles and mechanisms mediating social influences on daily timing. PMID:24500164

  16. Constant darkness restores entrainment to phase-delayed Siberian hamsters.

    PubMed

    Ruby, Norman F; Joshi, Nirav; Heller, H Craig

    2002-12-01

    Over 90% of Siberian hamsters (Phodopus sungorus) fail to reentrain to a 5-h phase delay of a 16:8-h photocycle. Because constant darkness (DD) restores rhythms disrupted by constant light, we tested whether DD could also restore entrainment. DD began 0, 5, or 14 days after a 5-h phase delay, and the light-dark cycle was reinstated 14 days later. All hamsters exposed to DD on day 0 reentrained, whereas 42% reentrained irrespective of whether DD began 5 or 14 days later. For these latter two groups, tau (tau) and alpha (alpha) in DD predicted reentrainment; animals that reentrained had a mean tau and alpha of 24.1 and 8.9 h, respectively, whereas those that failed to reentrain maintained a mean tau and alpha of 25.0 and of 7.1 h, respectively. Restoration of entrainment by DD is somewhat paradoxical because it suggests that reentrainment to the photocycle was prevented by continued exposure to that same photocycle. The dichotomy of circadian responses to DD suggests "entrainment" phenotypes that are similar to those of photoperiodic responders and nonresponders.

  17. Dose response of elastase-induced emphysema in hamsters.

    PubMed

    Raub, J A; Mercer, R R; Miller, F J; Graham, J A; O'Neil, J J

    1982-04-01

    Elastase-induced emphysema in hamsters was studied using pulmonary function tests in an effort to develop techniques for determining the effects of air pollutants on the progression of this disease. Single intratracheal injections of 6, 12, or 24 units of porcine pancreatic elastase produced dose-related changes in pulmonary function after 4 wk when compared with sham-injected control animals. Boyle's law end-expiratory volume and residual volume, measured by gas dilution, increased (p less than 0.05) at 12 and 24 units, respectively, whereas vital capacity, determined plethysmographically, and total lung capacity wee increased (p less than 0.05) at all 3 elastase doses. Respiratory system compliance, calculated by a nonlinear least squares regression fit of the deflation pressure-volume curve, increased (p less than 0.05) at 24 units only. The multiple-breath nitrogen washout slope (N2 slope) and the single-breath diffusing capacity for carbon monoxide (DLCO) decreased (p less than 0.05) at all 3 doses of elastase. Both histologic and physiologic evaluation showed dose-related pulmonary impairment. It appears, therefore, that as little as 6 units of elastase produces mild emphysema in hamsters, which is detectable by pulmonary function testing. Of these tests, the DLCO and N2 slope were the most effective in detecting the degree of impairment. PMID:6918202

  18. Rift Valley Fever Virus Infection in Golden Syrian Hamsters

    PubMed Central

    Scharton, Dionna; Van Wettere, Arnaud J.; Bailey, Kevin W.; Vest, Zachary; Westover, Jonna B.; Siddharthan, Venkatraman; Gowen, Brian B.

    2015-01-01

    Rift Valley fever virus (RVFV) is a formidable pathogen that causes severe disease and abortion in a variety of livestock species and a range of disease in humans that includes hemorrhagic fever, fulminant hepatitis, encephalitis and blindness. The natural transmission cycle involves mosquito vectors, but exposure can also occur through contact with infected fluids and tissues. The lack of approved antiviral therapies and vaccines for human use underlies the importance of small animal models for proof-of-concept efficacy studies. Several mouse and rat models of RVFV infection have been well characterized and provide useful systems for the study of certain aspects of pathogenesis, as well as antiviral drug and vaccine development. However, certain host-directed therapeutics may not act on mouse or rat pathways. Here, we describe the natural history of disease in golden Syrian hamsters challenged subcutaneously with the pathogenic ZH501 strain of RVFV. Peracute disease resulted in rapid lethality within 2 to 3 days of RVFV challenge. High titer viremia and substantial viral loads were observed in most tissues examined; however, histopathology and immunostaining for RVFV antigen were largely restricted to the liver. Acute hepatocellular necrosis associated with a strong presence of viral antigen in the hepatocytes indicates that fulminant hepatitis is the likely cause of mortality. Further studies to assess the susceptibility and disease progression following respiratory route exposure are warranted. The use of the hamsters to model RVFV infection is suitable for early stage antiviral drug and vaccine development studies. PMID:25607955

  19. Acid-induced secretory cell metaplasia in hamster bronchi

    SciTech Connect

    Christensen, T.G.; Lucey, E.C.; Breuer, R.; Snider, G.L.

    1988-02-01

    Hamsters were exposed to an intratracheal instillation of 0.5 ml of 0.08 N nitric, hydrochloric, or sulfuric acid to determine their airway epithelial response. Three weeks after exposure, the left intrapulmonary bronchi in Alcian blue/PAS-strained paraffin sections were evaluated for the amount of secretory product in the airway epithelium as a measure of secretory cell metaplasia (SCM). Compared to saline-treated control animals, all three acids caused statistically significant SCM. In addition to the bronchial lesion, all three acids caused similar interstitial fibrosis, bronchiolectasis, and bronchiolization of alveoli that varied in individual animals from mild to severe. In a separate experiment to study the persistence of the SCM, hamsters treated with a single instillation of 0.1 N nitric acid showed significant SCM 3, 7, and 17 weeks after exposure. There was a high correlation (r = 0.96) between a subjective assessment of SCM and objective assessment using a digital image-analysis system. We conclude that protons induce SCM independently of the associated anion; the SCM persists at least 17 weeks. Sulfuric acid is an atmospheric pollutant and nitric acid may form locally on the mucosa of lungs exposed to nitrogen dioxide. These acids may contribute to the development of maintenance of the SCM seen in the conducting airways of humans with chronic obstructive pulmonary disease.

  20. Sex differences in Siberian hamster ultradian locomotor rhythms.

    PubMed

    Prendergast, Brian J; Stevenson, Tyler J; Zucker, Irving

    2013-02-17

    Sex differences in ultradian activity rhythms (URs) and circadian rhythms (CRs) were assessed in Siberian hamsters kept in long day (LD) or short day (SD) photoperiods for 40 weeks. For both sexes URs of locomotor activity were more prevalent, greater in amplitude and more robust in SDs. The UR period was longer in females than males in both day lengths. The reproductive system underwent regression and body mass declined during the initial 10 weeks of SD treatment, and in both sexes these traits spontaneously reverted to the LD phenotype at or before 40 weeks in SD, reflecting the development of neuroendocrine refractoriness to SD patterns of melatonin secretion. Hamsters of both sexes, however, continued to display SD-like URs at the 40 weeks time point. CRs were less prevalent and the waveform less robust and lower in amplitude in SDs than LDs; the SD circadian waveform also did not revert to the long-day phenotype after 40 weeks of SD treatment. Short day lengths enhanced ultradian and diminished circadian rhythms in both sexes. Day length controls several UR characteristics via gonadal steroid and melatonin-independent mechanisms. Sex differences in ultradian timing may contribute to sex diphenisms in rhythms of sleep, food intake and exercise. PMID:23333554

  1. Effects of in vitro maturation of monkey oocytes on their developmental capacity

    PubMed Central

    Zheng, P.

    2007-01-01

    The study of in vitro maturation (IVM) of rhesus monkey oocytes has important implications for biomedical research and human infertility treatment. In vitro-matured rhesus monkey oocytes show much less developmental potential than IVM oocytes of other species. Since about 1980 when rhesus monkey IVM, in vitro fertilization (IVF) and in vitro embryo culture (IVC) systems were established, numerous efforts have been made to improve the developmental competence of oocytes and to understand the mechanisms regulating oocyte maturation. This review describes recent progress in this area, particularly the effects of factors such as steroid hormones, energy substrates, amino acids, ovarian follicle status, maternal age and breeding season on the developmental competence, gene expression patterns and genome integrity of rhesus IVM oocytes. PMID:17081707