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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 for 20-120 min and then microinjected into hamster oocytes to determine whether they could decondense and develop into pronucl...

  2. 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...

  3. Changes of spontaneous parthenogenetic activation and development potential of golden hamster oocytes during the aging process.

    PubMed

    Jiang, Han; Wang, Ce; Guan, Jiyu; Wang, Lingyan; Li, Ziyi

    2015-01-01

    The golden hamster is an excellent animal experimental model for oocyte research. The hamster oocytes are very useful in clinical examination of human spermatozoan activity. Non-fertile oocytes can lead to time-dependent processes of aging, which will affect the results of human spermatozoa examination. As a consequence there is a need to investigate the aging and anti-aging processes of golden hamster oocytes. In order to study the aging processes and parthenogenetic activation of golden hamster oocytes, in vivo oocytes, oocytes cultured with or without cumulus cells, and oocytes treated with Trichostatin A (TSA) or caffeine were collected and investigated. We found that: (1) spontaneous parthenogenetic activation, developmental potential (cleavage rate), and zona pellucida (ZP) hardening undergo age-dependent changes in in vivo, in vitro, and after TSA or caffeine treatment; (2) in vivo, oocytes became spontaneously parthenogenetic 25 h post-hCG treatment; (3) in vitro, cumulus cells did not significantly increase the parthenogenetic activation rate of cultured hamster oocytes; and (4) TSA or caffeine could delay spontaneous oocyte parthenogenetic activation and the aging processes by at least 5h, but also accelerated the hardening of the ZP. These results define the conditions for the aging and anti-aging processes in golden hamster oocytes. TSA and caffeine play roles in controlling spontaneous activation, which could facilitate the storage and use of golden hamster oocytes for studying processes relevant to human reproduction. PMID:25480399

  4. DNA (DEOXYRIBONUCLEIC ACID) SYNTHESIS FOLLOWING MICROINJECTION OF HETEROLOGOUS SPERM AND SOMATIC CELL NUCLEI INTO HAMSTER OOCYTES

    EPA Science Inventory

    The authors have 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 3H-thymidine after being parthenogenetically activated by sha...

  5. 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.

  6. Protamine withdrawal from human sperm nuclei following heterologous ICSI into hamster oocytes.

    PubMed

    Jones, Estella L; Zalensky, Andrei O; Zalenskaya, Irina A

    2011-08-01

    During late stages of spermatogenesis in mammals, most histones bound to DNA are replaced by protamines (PRM), which results in formation of supercondensed and genetically inert sperm chromatin. At fertilization, mature spermatozoon penetrates oocyte and chromatin is remodeled "back" from nucleoprotamine to nucleohistone state. While being crucial for activation of male genome and ultimately for initiation of embryonic development, this process is poorly studied, especially in humans. Data on model animals concerning PRM to histones exchange post fertilization are few and contradictory. As direct experimentation with human embryos is impossible due to ethical, legal and technical reasons, we evaluate the timing and mode of PRM removal in a heterologous ICSI system using hamster ova injected with human sperm. Localization of human PRM 1 and 2 in hybrid zygotes was established using immunofluorescence. We observed a marked zygote to zygote variability in male pronuclei size for any time point post ICSI and demonstrated that PRM removal correlates with the developing pronuclei area rather than time after injection. Overall, the disappearance of protamines from sperm is rather rapid and most likely completed within 1 hr. We propose that the critical characteristic influencing PRM removal after heterologous fertilization is the intrinsic heterogeneity of the human sperm population. The same yet unexplored variance may be one of the reasons for canceled, delayed or aberrant early embryonic development during natural or artificial fertilization in humans. PMID:21443492

  7. 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.

  8. Characterization of an oviductal glycoprotein associated with the ovulated hamster oocyte

    SciTech Connect

    Robitaille, G.; St-Jacques, S.; Potier, M.; Bleau, G.

    1988-04-01

    Hamster oviducts in culture incorporate (/sup 35/S)-methionine into secretory proteins. One of these proteins is immunoprecipitated by a monoclonal antibody specific to an antigen found in oviductal oocytes but not in ovarian oocytes. This antigen, called oviductin, is progressively added to the oocyte during its transit through the oviduct. Oviductin migrates as a diffuse band with a molecular mass between 160 and 250 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. The electrophoretic behavior of this protein suggests the presence of polysaccharide side chains. Chemical deglycosylation causes a decrease in molecular mass and removes the antigenic determinant originally present on the glycoprotein. By using the radiation inactivation method, the molecular mass of the core protein has been found to be approximately 44 kDa. These results indicate that the oviduct is an actual site of synthesis of the oviductin. This glycoprotein contains a high proportion of sugar residues, which account for antigenic determinant recognized by the monoclonal antibody.

  9. Zona pellucida resistance to sperm penetration before the completion of human oocyte maturation.

    PubMed

    Tesark, J; Pilka, L; Trvnk, P

    1988-05-01

    Human oocytes exposed to capacitated spermatozoa in vitro when at metaphase of the 1st meiotic division (metaphase I) were not penetrated, even though some subsequently progressed to metaphase of the 2nd meiotic division (metaphase II). When the non-penetrated oocytes that had reached metaphase II during the incubation with spermatozoa were freed from the zona pellucida and reinseminated, two or more pronuclei developed in most of them. By contrast, no penetration was observed when the oocytes were reinseminated in the zona-intact state. When compared with metaphase II oocytes, metaphase I oocytes had a similar zona-binding capacity for spermatozoa, but fewer spermatozoa were found within the zona. These results indicate that the zona pellucida of human oocytes undergoes important maturational changes during the transition from metaphase I to metaphase II. Ultrastructural and previous histochemical findings suggest that these changes involve secretions from both the oocyte and cumulus cells and that the increased zona resistance at metaphase I may be due to relative insufficiency of cumulus cell-secreted 'softening' factors. If the integrity of the cumulus oophorus is disturbed at this stage, this condition becomes irreversible. PMID:3397956

  10. Induction of aneuploidy in Chinese hamster oocytes following in vivo treatments with trimethoxybenzoic compounds and their analogues.

    PubMed

    Tateno, H; Kamiguchi, Y; Shimada, M; Sugawara, S; Mikamo, K

    1995-03-01

    Many inhibitors of tubulin polymerization have a trimethoxybenzene ring in their molecules. Such trimethoxybenzoic compounds and their analogues may therefore have a potency to induce meiotic nondisjunction of oocytes. In this study, a single dose of reserpine (0.5 microgram/g body weight), podophyllotoxin (20.0 micrograms/g b.w.), trimethoxybenzoic acid (500.0 micrograms/g b.w.) or vinblastine sulfate (3.0 micrograms/g b.w.) was injected intraperitoneally to mature female Chinese hamsters at the onset of the first meiotic spindle formation of oocytes. Within 6 h after spontaneous ovulation, MII oocytes were collected from the oviducts for morphological examination and cytogenetic analysis. The incidence of morphologically abnormal oocytes with unusually large first polar body or bodies increased significantly after the treatment with reserpine (18/202; 8.9%), podophyllotoxin (28/172; 16.3%) and vinblastine sulfate (63/197; 32.0%), as compared with the control (3/214; 1.4%). Chromosome analysis of oocytes revealed that podophyllotoxin and vinblastine sulfate were effective in inducing aneuploidy (62/154; 40.3% and 128/156; 82.1% vs. 3/198; 1.5% of the control) by inhibiting the formation of spindle microtubules at the first meiosis. Aneuploids were found more frequently in morphologically abnormal oocytes than in normal oocytes. No aneugenic activity of reserpine and trimethoxybenzoic acid was observed. These results indicate that trimethoxybenzoic compounds do not necessarily exhibit aneugenic activity. PMID:7870093

  11. Penetration of the zona pellucida of nonliving human oocytes by human spermatozoa in vitro.

    PubMed

    Overstreet, J W; Hembree, W C

    1976-07-01

    In an in vitro assay human spermatozoa penetrated the zona pellucida of 38.8% of 773 human oocytes recovered from the ovaries of cadavers. Zona penetration was not observed until 8 hours had elapsed. Oocytes examined with the electron microscope were surrounded mainly by sperm with intact acrosomes, but contained sperm in the zona and perivitelline space which had lost the outer acrosomal membrane and acrosomal contents. Sperm entry into the ooplasm was never observed. Spermatozoa from 11 of 16 patients with suspected infertility penetrated the zona, although the penetration rate was lower than that with sperm from fertile donors (12.9% versus 46.4%). When oocytes were incubated with mixed suspensions containing equal numbers of motile sperm from donors and patients, the donor sperm (identified by a fluorescent label) penetrated 50.0% and the patient sperm only 12.7%. These data suggest that human sperm penetrate the zona pellucida of nonliving human oocytes and mature living ova in a similar manner. This method is a potential diagnostic and investigative tool which avoids the ethical and technical problems associated with human in vitro fertilization. PMID:820576

  12. CARBENDAZIM (MBC) DISRUPTS OOCYTE SPINDLE FUNCTION AND INDUCES ANEUPLOIDY IN HAMSTERS EXPOSED DURING FERTILIZATION (MEIOSIS II)

    EPA Science Inventory

    Peri-fertilization exposure to Carbendazim (MBC; a microtubule poison) induces infertility and early pregnancy loss (EPL) in hamsters. resently, both in vivo and in vitro techniques were employed to characterize the effects of MBC on cellular aspects of fertilization in hamsters....

  13. 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. PMID:2776774

  14. 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 ?g/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

  15. Serial study of the effect of radiotherapy on semen parameters, hamster egg penetration rates, and lymphocyte chromosome abnormalities

    SciTech Connect

    Martin, R.H.; Barnes, M.; Arthur, K.; Ringrose, T.; Douglas, G.

    1984-02-01

    This study was designed to assess the long-term effects of radiotherapy (RT) on male fertility and the induction of lymphocyte and sperm chromosome abnormalities. This preliminary report provides information on 11 cancer patients (mainly seminomas) treated by RT (testicular dose, 44 to 499 rads). All 11 men were studied pre-RT and at intervals post-RT. The pre-RT semen profile varied considerably, but, in general, the profile was poor with a mean sperm concentration of 19.4 x 10/sup 6/ ml and a mean hamster egg penetration rate of 5%. One month after RT, the sperm concentration decreased and hamster egg penetration was 0% in all men. At 3 and 12 months post-RT, all but two patients were azoospermic. By 24 months post-RT, 9 of 11 patients had regained sperm production and 5 had sperm capable of hamster egg penetration. The three men who have been studied 36 months post-RT had a mean sperm concentration of 45.3 x 10/sup 6/ ml, and all had positive hamster egg penetration tests, although two of the three men had very low penetration rates (2% and 4%). Lymphocyte chromosome analysis demonstrated a striking frequency of chromosome abnormalities post-RT which decreased with time (pre-RT, 0%; 1 month, 42.4%; 3 months, 24.7%; 12 months, 13.8%; 24 months, 11.2%; and 36 months, 10.0%). Thus, it appears that sperm production starts to recover 2 to 3 years after RT when the frequency of lymphocyte chromosome abnormalities has decreased, but the sperm may not be fully functional at this time, as evidenced by poor rates of hamster egg penetration. Future studies of sperm chromosome analysis in these men will determine whether this impairment of the sperm is associated with meiotic chromosome abnormalities.

  16. In vitro and in vivo studies reveal that hamster oocyte meiotic arrest is maintained only transiently by follicular fluid, but persistently by membrana/cumulus granulosa cell contact.

    PubMed

    Racowsky, C; Baldwin, K V

    1989-08-01

    Studies were carried out with the golden Syrian hamster to investigate the capacity of follicular fluid to maintain oocyte meiotic arrest and to determine the importance of cumulus-membrana granulosa cell contact in the regulation of meiotic status. The follicular fluid studies were conducted by cytological assessment of meiotic stage up to 6 hr after transferring cumulus-free oocytes into antra of explanted "host" follicles in vitro or into follicles of anesthetized animals prior to the gonadotropin surge at proestrus in vivo. The cumulus-membrana granulosa contact studies were undertaken with explanted follicles in which the oocyte-cumulus complex was dislodged from the underlying membrana granulosa, released into the antrum, and subsequently allowed to reestablish contact during 6 hr of incubation within the follicle. The extent of recontact of the dislodged complex with the underlying membrana granulosa was assessed visually at the end of incubation and was classified as close, moderate, or none. These various degrees of contact typically involved the following number of cumulus cells, as determined by serial sectioning of a representative sample of follicles after dislodgement and subsequent incubation: close, 32.7 +/- 1.78; moderate, 9.0 +/- 2.1; and no contact, 0. After 6 hr of incubation either in vitro or in vivo, few transferred oocytes remained at the germinal vesicle (GV) stage (18.8 +/- 8.7 and 17.3 +/- 4.0% GV, respectively). However, time course experiments revealed that meiotic resumption was significantly delayed in transferred oocytes compared with either liberated oocytes, spontaneously maturing oocytes, or follicle-enclosed oocytes induced to mature by luteinizing hormone in vitro (after 4 hr, transferred, 31.3 +/- 6.0% GV; liberated, 0% GV; follicle-enclosed, 0% GV; after 6 hr, 0% transferred oocytes exhibited a GV). In the dislodgement studies, after 6 hr of incubation, 26% of complexes reestablished close contact with the underlying membrana granulosa, 67% showed moderate contact, while 7% revealed no contact. There was a significant increase in the percentage GV stage oocytes as the extent of recontact increased (no contact, 21.9 +/- 3.6% GV; moderate contact, 56.6 +/- 6.8% GV; close contact, 87.5 +/- 14.4% GV). These data argue in favor of a stringent control of hamster oocyte meiotic status by the follicle cell/oocyte syncytium and against the possibility that follicular fluid is independently responsible for maintaining meiotic arrest.(ABSTRACT TRUNCATED AT 400 WORDS) PMID:2744234

  17. 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 surfaces of individual sperm. An effective viscosity of the cumulus near its border was estimated to be of the order of 1-4 P. PMID:3220430

  18. 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. PMID:26443234

  19. IMPORTANCE OF GLUTATHIONE IN THE ACQUISITION AND MAINTENANCE OF SPERM NUCLEAR DECONDENSING ACTIVITY IN MATURING HAMSTER OOCYTES

    EPA Science Inventory

    Sperm nuclear decondensing activity in mammalian oocytes is dependent upon the maturational state of the oocyte. It is maximal in mature, metaphase II oocytes and minimal or absent in immature germinal vesicle (GV) and fertilized pronuclear oocytes. Previous studies suggested tha...

  20. 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

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

    PubMed Central

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

    2014-01-01

    ABSTRACT The objective of this research was to estimate the optimal timing for fertilization to achieve proper embryonic development of in vitro-matured bovine oocytes. First, cumulus-oocyte complexes were subjected to in vitro maturation (IVM) for 1422 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 1230 hr, sperm penetration was examined after 418 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 1230 hr of IVM were inseminated and cultured for 6 days to examine embryonic development. In the group with 22 hr of IVM, the percentages of cleaved embryos and blastocysts were the highest values in all groups. According to the regression equation describing the time from nuclear maturation to sperm penetration (x) and the percentage of blastocysts (y) (y=7.23x ? 0.297x2, P<0.01), the blastocyst rate peaked when sperm penetration occurred at 12.2 hr after achieving nuclear maturation. In conclusion, under the present IVM/IVF conditions, it was estimated that oocytes acquired their highest developmental competence at about 30 hr after IVM start, and thus, the optimal IVM duration was calculated to be about 21 hr. PMID:24430663

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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 oocytes and decline after fertiliz...

  3. Zona pellucida from fertilised human oocytes induces a voltage-dependent calcium influx and the acrosome reaction in spermatozoa, but cannot be penetrated by sperm

    PubMed Central

    Patrat, Catherine; Auer, Jana; Fauque, Patricia; Leandri, Roger L; Jouannet, Pierre; Serres, Catherine

    2006-01-01

    Background The functions of three zona glycoproteins, ZP1, ZP2 and ZP3 during the sperm-zona pellucida (ZP) interaction are now well established in mice. The expression of an additional zona glycoprotein, ZPB/4, in humans, led us to reconsider the classical mouse model of gamete interaction. We investigated the various functions of human ZP (hZP) during the interaction of spermatozoa with fertilised and unfertilised oocytes. Results The hZP of fertilised oocytes retained their ability to bind sperm (albeit less strongly than that from unfertilised oocytes), to induce an intraspermatic calcium influx through voltage-dependent channels similar to that observed with hZP from unfertilised oocytes and to promote the acrosome reaction at a rate similar to that induced by the ZP of unfertilised oocytes (61.6 6.2% vs60.7 9.1% respectively). Conversely, the rate of hZP penetrated by sperm was much lower for fertilised than for unfertilised oocytes (19% vs 57% respectively, p < 0.01). We investigated the status of ZP2 in the oocytes used in the functional tests, and demonstrated that sperm binding and acrosome reaction induction, but not ZP penetration, occurred whether or not ZP2 was cleaved. Conclusion The change in ZP function induced by fertilisation could be different in human and mouse species. Our results suggest a zona blocking to polyspermy based at the sperm penetration level in humans. PMID:17147816

  4. 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...

  5. Efficacy of zona-free hamster egg sperm penetration assay as a predictor of in vitro fertilization.

    PubMed

    Ibrahim, M E; Moussa, M A; Pedersen, H

    1989-01-01

    The efficacy of the zona-free hamster egg sperm penetration assay (SPA) as a predictor of in vitro fertilization (IVF) is studied. Indications for IVF were tubal factors in 35 couples and male factors in 24 couples. The diagnostic characteristics of SPA in reference to IVF for the whole group (n = 59) were reasonably reliable. Sensitivity was 74%, specificity was 84%, and diagnostic accuracy was 81%. However in patients with male factors, SPA was less reliable in terms of sensitivity (70%), specificity (57%), and diagnostic accuracy (63%), than in patients with tubal factors where the indicators were 80%, 97%, and 94%, respectively. Semen from patients with male infertility shared notable variations in terms of sperm density and motility which may account for the low SPA predictivity. SPA seems to be a useful screening test for prediction of IVF outcome. However other semen variables should be considered before interpretation, especially in male infertility where the diagnostic accuracy is low. PMID:2619415

  6. 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 assessment of morphology may be an unreliable measure, for the individual, of sperm fertilizing ability and emphasize that sperm function testing is an important part of the evaluation of teratospermia. PMID:17460097

  7. Pre-pubertal di(2-ethylhexyl) phthalate (DEHP) exposure of young boars did not affect sperm in vitro penetration capacity of homologous oocytes post-puberty.

    PubMed

    Spjuth, Linda; Gil, M A; Caballero, I; Cuello, C; Almiana, C; Martnez, E A; Lundeheim, N; Rodrguez-Martnez, H

    2007-01-01

    Di(2-ethylhexyl) phthalate (DEHP), a plastic softener used in polyvinylchloride (PVC) products (e.g., plastic bags and medical equipment), has been reported to have toxic effects on animal reproduction and is considered an environmental hazard based, mostly, on rodent studies. However, the doses used in these studies are often considerably higher than that presumed in human exposure. In the present study we used young boars as model animals to assess the effects of pre-pubertal DEHP exposure on the ability of spermatozoa to penetrate homologous oocytes in vitro. Eight pairs of cross-bred male boar siblings were used. One brother in each pair became, at random, the test animal exposed to DEHP per os, three times a week, from 3 to 7 weeks of age while the other acted as the control, i.e., placebo-exposed. Semen was collected and frozen between 8 and 9 months of age and stored until spermatozoa were evaluated for their ability to in vitro penetrate in vitro-matured homologous oocytes post-thaw. Both the penetration rate and the number of spermatozoa per oocyte were considered within expected ranges for frozen boar semen of good quality. Penetration rate did not significantly differ (p > 0.05) between the groups with DEHP-exposed: 50%; control: 59%, which could be owing to a large variation between boars, and between replicates. The number of spermatozoa in the ooplasm was low and similar (p > 0.05) between the groups with DEHP-exposed: 1.5 and the control: 1.7. Under the conditions of the present experiment, pre-pubertal exposure to DEHP does not seem to cause a deleterious effect on the in vitro fertilizing ability of frozen spermatozoa post-puberty. PMID:17612872

  8. 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...

  9. 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.

  10. [Oocyte cryopreservation].

    PubMed

    Hourvitz, Ariel; Maman, Ettie; Meiri-Farber, Betty; Dor, Joshua

    2008-02-01

    Oocyte cryopreservation is an important method in the field of infertility. This procedure can benefit the cancer patient wishing to preserve fertility before initiation of any destructive chemotherapy or radiation therapy. It is a substitute for embryo cryopreservation and thereby avoids associated ethical issues. Oocyte cryopreservation technology can lead to the establishment of "oocytes banks" and provides solutions to ovarian failure patients. Technical obstacles were the main cause for the slow progress of oocyte cryopreservation. When compared to embryos and sperm cryopreservation technologies which are in common use worldwide, the reports on oocyte cryopreservation-derived pregnancies, were sporadic and with low success rates. However, in recent years we are witnessing more and more publications and increasing success rates in this important and fascinating field. Improvement in the freezing protocols and the introduction of vitrification procedures increased the survival rates and led to the birth of more then 100 healthy newborns. In the future we believe this method will be part of daily work in the fertility world. This review describes the methods available today and the clinical reports published in this field. PMID:18357674

  11. [In vitro maturation of human oocytes].

    PubMed

    Plachot, M

    1999-06-01

    Maturation of human oocytes has 3 aspects: nuclear maturation leading to the extrusion of the 1st polar body, membranar maturation essential for the fixation of spermatozoa to the zona pellucida and penetration into the oocyte and cytoplasmic maturation which allows protein synthesis required for normal fertilisation and embryo development. In vitro maturation (IVM) of human oocytes may be appropriate in 5 different situations: for PCOs patients (natural cycle), in normoovulatory patients (natural cycles), for oocytes not exposed to hCG (stimulated cycle), for immature oocytes recovered in the course of an ICSI protocol (stimulated cycle) and after freezing-thawing of immature oocytes. Data from the literature show that in vitro maturation of human oocytes together with ICSI can lead to normal fertilisation, embryo development, pregnancies and the delivery of healthy children. However, the overall efficiency is still very low, indicating that embryo viability is compromised. The incidence of chromosome abnormalities in mature oocytes obtained after IVM is similar to that of oocytes recovered after in vivo maturation and therefore does not seem to be the reason of the failures. Conversely, protein synthesis abnormalities and abnormal calcium signalling might explain the poor viability of the embryos. The key factor seems to be cytoplasmic maturation not yet fully understood in the human. PMID:10431449

  12. Evaluation of Zona Pellucida Function for Sperm Penetration During In Vitro Fertilization in Pigs

    PubMed Central

    TANIHARA, Fuminori; NAKAI, Michiko; KANEKO, Hiroyuki; NOGUCHI, Junko; OTOI, Takeshige; KIKUCHI, Kazuhiro

    2013-01-01

    Abstract In porcine oocytes, the function of the zona pellucida (ZP) with regard to sperm penetration or prevention of polyspermy is not well understood. In the present study, we investigated the effects of the ZP on sperm penetration during in vitro fertilization (IVF). We collected in vitro-matured oocytes with a first polar body (ZP+ oocytes). Some of them were freed from the ZP (ZP− oocytes) by two treatments (pronase and mechanical pipetting), and the effects of these treatments on sperm penetration parameters (sperm penetration rate and numbers of penetrated sperm per oocyte) were evaluated. There was no evident difference in the parameters between the two groups. Secondly, we compared the sperm penetration parameters of ZP+ and ZP− oocytes using frozen-thawed epididymal spermatozoa from four boars. Sperm penetration into ZP+ oocytes was found to be accelerated relative to ZP− oocytes. Thirdly, we evaluated the sperm penetration of ZP+ and ZP− oocytes at 1−10 h after IVF (3 h gamete co-incubation). The proportions of oocytes penetrated by sperm increased significantly with time in both groups; however, the number of penetrated sperm per oocyte did not increase in ZP− oocytes. Finally, we performed IVF using ZP− oocytes divided into control (3 h) and prolonged gamete co-incubation (5 h) groups. Greater numbers of sperm penetrated in the 5 h group than in the control group. These results suggest that the ZP and oolemma are not competent factors for prevention of polyspermy in our present porcine IVF system. However, it appears that ZP removal is one of the possibilities for reducing polyspermic penetration in vitro in pigs. PMID:23666494

  13. Chromosomal abnormalities in oocytes.

    PubMed

    Plachot, M

    2001-10-22

    Since the beginning of in vitro fertilization (IVF), basic research has provided insight in the field of human reproduction, especially in genetics. Indeed, the contribution of chromosomal abnormalities to oocyte disorders and impaired embryonic development is now well known. Of oocytes that fail to fertilize after in vitro insemination, 26.5% have been found to be abnormal, with 13.3% showing hypohaploidy, 8.1% hyperhaploidy, 1.6% structural abnormalities and 3.5% diploidy. The total incidence of abnormalities seems to be correlated with the fertility status of the woman. It is higher in oocytes from women with tubal or unexplained infertility than in those from women whose husband's infertility is the sole cause of infertility in the couple. Although few oocytes recovered during natural cycles have been studied, gonadotropins, which are widely used to stimulate follicle growth and ovulation, do not increase the risk of abnormalities. The effect of maternal age on fetal aneuploidy, well documented at birth, has not been unambiguously shown to result from an increase in the frequency of aneuploid oocytes. Intra- and extra-follicular influences (perifollicular microvasculature, oxygenation, and the presence of residues from cigarette smoke) may disturb maturation, leading to immaturity and aneuploidy. Thus, oocyte meiosis is very sensitive to endogenous and exogenous factors that could result in oocytes with chromosomal abnormalities and therefore, abnormal zygotes. PMID:11576735

  14. Nuclear transfer and oocyte cryopreservation.

    PubMed

    Chang, Ching-Chien; Sung, Li-Ying; Amano, Tomokazu; Tian, X Cindy; Yang, Xiangzhong; Nagy, Zsolt Peter

    2009-01-01

    Somatic cells can be reprogrammed to a totipotent state through nuclear transfer or cloning, because it has been demonstrated that the oocyte has the ability to reprogramme an adult nucleus into an embryonic state that can initiate the development of a new organism. Therapeutic cloning, whereby nuclear transfer is used to derive patient-specific embryonic stem cells, embraces an entire new opportunity for regenerative medicine. However, a key obstacle for human therapeutic cloning is that the source of fresh human oocytes is extremely limited. In the present review, we propose prospective sources of human oocytes by using oocyte cryopreservation, such as an oocyte bank and immature oocytes. We also address some potential issues associated with nuclear transfer when using cryopreserved oocytes. In the future, if the efficacy and efficiency of cryopreserved oocytes are comparable to those of fresh oocytes in human therapeutic cloning, the use of cryopreserved oocytes would be invaluable and generate a great impact to regenerative medicine. PMID:19152744

  15. TIMING OF HAMSTER SPERM NUCLEAR DECONDENSATION AND MALE PRONUCLEUS FORMATION IS RELATED TO SPERM NUCLEAR DISULFIDE BOND CONTENT

    EPA Science Inventory

    The relationship between the timing of both sperm nuclear decondensation and male pronucleus formation in the oocyte and the relative level of disulfide bonds within the sperm nucleus was evaluated. Four types of hamster sperm nuclei, in which the extent of S-S bonding differed, ...

  16. Oocyte ageing and epigenetics.

    PubMed

    Ge, Zhao-Jia; Schatten, Heide; Zhang, Cui-Lian; Sun, Qing-Yuan

    2015-03-01

    It has become a current social trend for women to delay childbearing. However, the quality of oocytes from older females is compromised and the pregnancy rate of older women is lower. With the increased rate of delayed childbearing, it is becoming more and more crucial to understand the mechanisms underlying the compromised quality of oocytes from older women, including mitochondrial dysfunctions, aneuploidy and epigenetic changes. Establishing proper epigenetic modifications during oogenesis and early embryo development is an important aspect in reproduction. The reprogramming process may be influenced by external and internal factors that result in improper epigenetic changes in germ cells. Furthermore, germ cell epigenetic changes might be inherited by the next generations. In this review, we briefly summarise the effects of ageing on oocyte quality. We focus on discussing the relationship between ageing and epigenetic modifications, highlighting the epigenetic changes in oocytes from advanced-age females and in post-ovulatory aged oocytes as well as the possible underlying mechanisms. PMID:25391845

  17. 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

  18. Single oocyte bisulfite mutagenesis.

    PubMed

    Denomme, Michelle M; Zhang, Liyue; Mann, Mellissa R W

    2012-01-01

    Epigenetics encompasses all heritable and reversible modifications to chromatin that alter gene accessibility, and thus are the primary mechanisms for regulating gene transcription. DNA methylation is an epigenetic modification that acts predominantly as a repressive mark. Through the covalent addition of a methyl group onto cytosines in CpG dinucleotides, it can recruit additional repressive proteins and histone modifications to initiate processes involved in condensing chromatin and silencing genes. DNA methylation is essential for normal development as it plays a critical role in developmental programming, cell differentiation, repression of retroviral elements, X-chromosome inactivation and genomic imprinting. One of the most powerful methods for DNA methylation analysis is bisulfite mutagenesis. Sodium bisulfite is a DNA mutagen that deaminates cytosines into uracils. Following PCR amplification and sequencing, these conversion events are detected as thymines. Methylated cytosines are protected from deamination and thus remain as cytosines, enabling identification of DNA methylation at the individual nucleotide level. Development of the bisulfite mutagenesis assay has advanced from those originally reported towards ones that are more sensitive and reproducible. One key advancement was embedding smaller amounts of DNA in an agarose bead, thereby protecting DNA from the harsh bisulfite treatment. This enabled methylation analysis to be performed on pools of oocytes and blastocyst-stage embryos. The most sophisticated bisulfite mutagenesis protocol to date is for individual blastocyst-stage embryos. However, since blastocysts have on average 64 cells (containing 120-720 pg of genomic DNA), this method is not efficacious for methylation studies on individual oocytes or cleavage-stage embryos. Taking clues from agarose embedding of minute DNA amounts including oocytes, here we present a method whereby oocytes are directly embedded in an agarose and lysis solution bead immediately following retrieval and removal of the zona pellucida from the oocyte. This enables us to bypass the two main challenges of single oocyte bisulfite mutagenesis: protecting a minute amount of DNA from degradation, and subsequent loss during the numerous protocol steps. Importantly, as data are obtained from single oocytes, the issue of PCR bias within pools is eliminated. Furthermore, inadvertent cumulus cell contamination is detectable by this method since any sample with more than one methylation pattern may be excluded from analysis. This protocol provides an improved method for successful and reproducible analyses of DNA methylation at the single-cell level and is ideally suited for individual oocytes as well as cleavage-stage embryos. PMID:22782232

  19. 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

  20. Fertilization Ability of Porcine Oocytes Reconstructed from Ooplasmic Fragments Produced and Characterized after Serial Centrifugations

    PubMed Central

    Viet LINH, Nguyen; KIKUCHI, Kazuhiro; NAKAI, Michiko; TANIHARA, Fuminori; NOGUCHI, Junko; KANEKO, Hiroyuki; DANG-NGUYEN, Thanh Quang; MEN, Nguyen Thi; VAN HANH, Nguyen; SOMFAI, Tamas; NGUYEN, Bui Xuan; NAGAI, Takashi; MANABE, Noboru

    2013-01-01

    Abstract Mitochondria are reported to be critical in in vitro maturation of oocytes and subsequent embryo development after fertilization, but their contribution for fertilization has not been investigated in detail. In the present study, we investigate the contribution of mitochondria to fertilization using reconstructed porcine oocytes by fusion of ooplasmic fragments produced by serial centrifugations (centri-fusion). Firstly, we evaluated the characteristics of ooplasmic fragments. Three types of fragments were obtained by centrifugation of porcine oocytes matured in vitro for 46 h: brownish (B), transparent (T) and large (L) fragments containing both B and T parts in a fragment. The production efficiencies of these types of fragments were 71.7, 91.0 and 17.8 fragments/100 oocytes, respectively. In experiments, L fragments were excluded because they contained both brownish and transparent components that were apparently intermediate between B and T fragments. Observations by confocal microscopy after staining with MitoTracker Red CMXRos and transmission electron microscopy revealed highly condensed active mitochondria in B fragments in contrast to T fragments that contained only sparse organelles. We reconstructed oocytes by fusion of a karyoplast and two cytoplasts from B and T fragments (B and T oocytes, respectively). The B oocytes showed higher sperm penetration (95.8%) and male pronuclear formation rates (94.2%) by in vitro fertilization than T oocytes (66.7% and 50.0%, respectively). These results suggest that the active mitochondria in oocytes may be related to their ability for fertilization. PMID:23965685

  1. Polyribosomes of Hamster Cells

    PubMed Central

    Stanners, Clifford P.

    1968-01-01

    The amount of radioactivity incorporated into completed polypeptides and into nascent polypeptides bound to polyribosomes was measured for rapidly dividing, cultured hamster embryo cells incubated in the presence of labelled amino acids. Using a mathematical analysis which took into account variations in the specific activity of the intracellular amino acid pool, these measurements yielded a value for the length of time required for the synthesis of a primary polypeptide of average length, the transit time. Subject to a number of reasonable assumptions in the mathematical analysis which were, however, only approximately verified, the transit time was found to be 23 2 sec in four independent experiments. Regardless of the absolute accuracy of this measurement, the method is considered a useful measure of translation independent of transcription. PMID:5689103

  2. Meiotic progression, mitochondrial features and fertilisation characteristics of porcine oocytes with different G6PDH activities.

    PubMed

    Egerszegi, Istvn; Alm, Hannelore; Rtky, Jzsef; Heleil, Bassiouni; Brssow, Klaus-Peter; Torner, Helmut

    2010-01-01

    The aim of the present study was to investigate the developmental competence, mitochondrial characteristics and chromatin status of immature follicular porcine oocytes selected for their glucose-6-phosphate dehydrogenase (G6PDH) activity by brilliant cresyl blue (BCB) staining. In Experiment 1, the oocyte parameters were determined in parallel right after BCB staining (T(0)), after 22 h of in vitro maturation (IVM) (T(22)) and after 44 h of IVM (T(44)) (n = 496). BCB-stained oocytes (BCB+) at T(0) were characterised by fibrillated chromatin filaments in their germinal vesicles (GV) and diakinesis stages whereas unstained (BCB-) oocytes at T(0) contained in their GV mainly condensed stages of chromatin (P < 0.05). After 22 h of IVM BCB+ oocytes showed a prominent chromatin configuration of metaphase I and after 44 h the majority developed a M II nuclear configuration in contrast to the BCB- group (P < 0.0001). Differences were also observed between the two oocyte populations in their mitochondrial activity (P < 0.05). At the beginning of IVM BCB+ oocytes were characterised by high mitochondrial activity in their cytoplasm. The BCB+ oocytes showed clear visible homogenous distributions of mitochondria (P < 0.005) and contained more aggregated clusters of mitochondria in contrast to BCB- oocytes (P < 0.005). In Experiment 2, 318 oocytes were tested for their G6PDH activity and introduced to IVM and IVF. Only oocytes from the BCB+ group, which were matured after 44 h up to the stage of M II (81.6%) were fertilised (17.4%), penetrated (46%) or activated (15.6%) after IVF. These results indicate a relationship between the G6PDH activity of porcine oocytes before IVM and their subsequent nuclear development, mitochondrial activity and aggregation. PMID:20450835

  3. Sperm contributions to oocyte activation: more that meets the eye.

    PubMed

    Anifandis, George; Messini, Christina I; Dafopoulos, Konstantinos; Daponte, Alexandros; Messinis, Ioannis E

    2016-03-01

    It is well known that for successful fertilization, oocyte activation is required, which involves a signal transduction cascade leading to the conversion of the oocyte to a diploid embryo. During oocyte activation, intracellular calcium levels oscillate repetitively causing exocytosis of cortical granules, the enzymes which the latter contain are released into the perivitelline space, leading to modifications of the zona pellucida (ZP), which prevent the penetration of the ZP by further spermatozoa. Τhe necessary element that initiates oocyte activation is apparently the release of intracellular calcium (Ca(2+)) stored in the endoplasmic reticulum (ER). The exact mechanism via which Ca(2+) is released within the oocyte has not been yet clarified, and has been a matter of an ongoing debate. Today, the sperm factor hypothesis has gained general acceptance, according to which a sperm molecule, either phospholipase C (PLCζ) or a post-acrosomal sheath WW domain-binding protein (PAWP), diffuses into the ooplasm initiating a molecular cascade involving mainly the phosphoinositide pathway. Mounting evidence now indicates that these calcium oscillations are caused by a testis-specific PLC termed PLCζ, released into the oocyte following gamete fusion. Also, recently, PAWP has been proposed as an alternative sperm factor candidate. These different sperm candidates have led to a significant debate. This raises important questions as regards to the relative importance of these two proteins as diagnostic tools in reproductive medicine with therapeutic potential, indicating the need for further research. In the present mini review, the phenomenon of oocyte activation during fertilization as well as the existing controversy will be highlighted and the possible mechanisms that are involved in this process will be discussed. Finally, an explanation of the existing debate will be attempted. PMID:26780328

  4. The control of reactive oxygen species influences porcine oocyte in vitro maturation.

    PubMed

    Alvarez, G M; Morado, S A; Soto, M P; Dalvit, G C; Cetica, P D

    2015-04-01

    The aim of this study was to examine the effect of varying intracellular reactive oxygen species (ROS) levels during oocyte in vitro maturation with enzymatic ROS production systems (xanthine + xanthine oxidase or xanthine + xanthine oxidase + catalase), scavenger systems (catalase or superoxide dismutase + catalase) or cysteine on porcine oocyte maturation. Oocyte ROS levels showed an increase when H2O2 or O2?(-) production systems were added to the culture medium (p < 0.05). On the other hand, the presence of ROS scavengers in the maturation medium did not modify oocyte ROS levels compared with the control after 48 h of maturation, but the addition of cysteine induced a decrease in oocyte ROS levels (p < 0.05). The ROS production systems used in this work did not modified the percentage of oocyte nuclear maturation, but increased the decondensation of sperm head (p < 0.05) and decreased the pronuclear formation (p < 0.05). In turn, the addition of O2?(-) and H2O2 scavenging systems during in vitro maturation did not modify the percentage of oocytes reaching metaphase II nor the oocytes with decondensed sperm head or pronuclei after fertilization. However, both parameters increased in the presence of cysteine (p < 0.05). The exogenous generation of O2?(-) and H2O2 during oocyte in vitro maturation would not affect nuclear maturation or later sperm penetration, but most of the spermatozoa cannot progress to form the pronuclei after fusion with the oocyte. The decrease in endogenous ROS levels by the addition of cysteine would improve pronuclear formation after sperm penetration. PMID:25522082

  5. Molecular and Cellular Mechanisms of Sperm-Oocyte Interactions Opinions Relative to in Vitro Fertilization (IVF)

    PubMed Central

    Anifandis, George; Messini, Christina; Dafopoulos, Konstantinos; Sotiriou, Sotiris; Messinis, Ioannis

    2014-01-01

    One of the biggest prerequisites for pregnancy is the fertilization step, where a human haploid spermatozoon interacts and penetrates one haploid oocyte in order to produce the diploid zygote. Although fertilization is defined by the presence of two pronuclei and the extraction of the second polar body the process itself requires preparation of both gametes for fertilization to take place at a specific time. These preparations include a number of consecutive biochemical and molecular events with the help of specific molecules and with the consequential interaction between the two gametes. These events take place at three different levels and in a precise order, where the moving spermatozoon penetrates (a) the outer vestments of the oocyte, known as the cumulus cell layer; (b) the zona pellucida (ZP); where exocytosis of the acrosome contents take place and (c) direct interaction of the spermatozoon with the plasma membrane of the oocyte, which involves a firm adhesion of the head of the spermatozoon with the oocyte plasma membrane that culminates with the fusion of both sperm and oocyte membranes (Part I). After the above interactions, a cascade of molecular signal transductions is initiated which results in oocyte activation. Soon after the entry of the first spermatozoon into the oocyte and oocyte activation, the oocyte’s coat (the ZP) and the oocyte’s plasma membrane seem to change quickly in order to initiate a fast block to a second spermatozoon (Part II). Sometimes, two spermatozoa fuse with one oocyte, an incidence of 1%–2%, resulting in polyploid fetuses that account for up to 10%–20% of spontaneously aborted human conceptuses. The present review aims to focus on the first part of the human sperm and oocyte interactions, emphasizing the latest molecular and cellular mechanisms controlling this process. PMID:25054321

  6. 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.

  7. Maturation of Oocytes in Vitro.

    PubMed

    Lonergan, Patrick; Fair, Trudee

    2016-02-15

    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. PMID:26566159

  8. Microinjection of Xenopus Laevis Oocytes

    PubMed Central

    Cohen, Sarah; Au, Shelly; Pant, Nelly

    2009-01-01

    Microinjection of Xenopus laevis oocytes followed by thin-sectioning electron microscopy (EM) is an excellent system for studying nucleocytoplasmic transport. Because of its large nucleus and high density of nuclear pore complexes (NPCs), nuclear transport can be easily visualized in the Xenopus oocyte. Much insight into the mechanisms of nuclear import and export has been gained through use of this system (reviewed by Pant, 2006). In addition, we have used microinjection of Xenopus oocytes to dissect the nuclear import pathways of several viruses that replicate in the host nucleus. Here we demonstrate the cytoplasmic microinjection of Xenopus oocytes with a nuclear import substrate. We also show preparation of the injected oocytes for visualization by thin-sectioning EM, including dissection, dehydration, and embedding of the oocytes into an epoxy embedding resin. Finally, we provide representative results for oocytes that have been microinjected with the capsid of the baculovirus Autographa californica nucleopolyhedrovirus (AcMNPV) or the parvovirus Minute Virus of Mice (MVM), and discuss potential applications of the technique. PMID:19238134

  9. Chromosome transfer in mature oocytes.

    PubMed

    Tachibana, Masahito; Sparman, Michelle; Mitalipov, Shoukhrat

    2010-06-01

    In this article, we describe detailed protocols for the isolation and transfer of spindle-chromosomal complexes between mature, metaphase II-arrested oocytes. In brief, the spindle-chromosomal complex is visualized using a polarized microscope and extracted into a membrane-enclosed karyoplast. Chromosomes are then reintroduced into an enucleated recipient egg (cytoplast), derived from another female, by karyoplast-cytoplast membrane fusion. Newly reconstructed oocytes consist of nuclear genetic material from one female and cytoplasmic components, including mitochondria and mitochondrial DNA (mtDNA), from another female. This approach yields developmentally competent oocytes suitable for fertilization and producing embryonic stem cells or healthy offspring. The protocol was initially developed for monkey oocytes but can also be used in other species, including mouse and human oocytes. Potential clinical applications include mitochondrial gene replacement therapy to prevent transmission of mtDNA mutations and treatment of infertility caused by cytoplasmic defects in oocytes. Chromosome transfer between the cohorts of oocytes isolated from two females can be completed within 2 h. PMID:20539289

  10. The human oocyte. Genetic aspects.

    PubMed

    Plachot, M

    1997-01-01

    Since the beginning of in vitro fertilization (IVF), basic research has enlightened the field of human reproduction, especially in genetics. Indeed, the contribution of chromosomal anomalies to oocyte disorders and impaired developmental capacities of the embryos is now well known. Among oocytes that failed to fertilize after in vitro insemination, 26.5% were found to be abnormal comprising 13.3% hypohaploidy, 8.1% hyperhaploidy, 1.6% structural anomalies and 3.5% diploidy. The total incidence of abnormalities seems to be correlated to the female status, and was found to be higher in oocytes from women with tubal or unexplained infertility than in those from women whose husband was infertile as a sole cause of couple infertility. Although few oocytes recovered during a natural cycle were studied, gonadotropins widely used to stimulate follicle growth and ovulation do not increase the risk of anomalies. The effect of maternal age on fetal aneuploidy, well documented at birth, has not yet be found unambiguously to be a consequence of an increased rate of aneuploid oocytes. Intra- and extrafollicular influences (perifollicular microvasculature, oxygenation, the presence of residues from cigarette smoke) are able to disturb maturation leading to immaturity and aneuploidy. To conclude, oocyte meiosis is very sensitive to endogenous or exogenous factors, which could lead to chromosomally abnormal oocytes and as a consequence, to abnormal zygotes. PMID:9259959

  11. 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.

  12. 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

  13. Hyperadrenocorticism in three teddy bear hamsters.

    PubMed

    Bauck, L B; Orr, J P; Lawrence, K H

    1984-06-01

    Hyperadrenocorticism was diagnosed in three related teddy bear hamsters with presenting complaints of alopecia and hyperpigmentation of the skin. Treatment was attempted in two of the hamsters and was successful in one case. Metyrapone and o,p'-DDD (1,1-dichloro-2-2bis (p-chlorophenyl) ethane) were the drugs used. Necropsy and histopathological examinations revealed a pituitary chromophobe adenoma in one hamster and an adrenocortical adenocarcinoma in a second hamster. The third related hamster was clinically diagnosed as having hyperadrenocorticism but the origin of the disease has not yet been determined. PMID:17422415

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

    PubMed Central

    Kabithe, Esther W.; Place, Ned J.

    2008-01-01

    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 if 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 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, which 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. PMID:18299426

  15. FLB1, a human protein of epididymal origin that is involved in the sperm-oocyte recognition process.

    PubMed

    Bou, F; Duquenne, C; Lassalle, B; Lefvre, A; Finaz, C

    1995-02-01

    CA6 antibody was selected out of a monoclonal antibody library raised against human sperm proteins primarily for its ability to recognize an epididymal antigen and to modify sperm adhesion to zona-free hamster oocytes. In the present study, CA6 was shown to decrease sperm binding to zona-free hamster and human oocytes by 40-92% and 38-48%, respectively. The corresponding protein, which was referred to as FLB1, was found to be secreted by the epididymis and to bind specifically to a human, macaque, and rodent subacrosomal sperm region. Western blotting revealed a molecular mass of 94 kDa in human epididymal extracts and of 100 kDa in human, macaque, mouse, rat, and hamster sperm, suggesting further modifications after its binding to sperm. An equivalent protein was not observed in human liver, ovary, testis, plasma, or epidermis. Two-dimensional electrophoresis showed that FLB1 is formed of two subunits with the same 47-kDa molecular mass and slightly different pI (5.8, 5.9). Microsequencing of the protein revealed a partial homology with human cytokeratins 1 and 10. These results suggest that FLB1 is an epididymis-specific cytokeratin-like protein that is involved in the sperm-oocyte recognition process. PMID:7536047

  16. 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. PMID:26454098

  17. 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 24% 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

  18. 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. PMID:21838965

  19. Penetrating trauma

    PubMed Central

    Kuhajda, Ivan; Zarogoulidis, Konstantinos; Kougioumtzi, Ioanna; Huang, Haidong; Li, Qiang; Dryllis, Georgios; Kioumis, Ioannis; Pitsiou, Georgia; Machairiotis, Nikolaos; Katsikogiannis, Nikolaos; Papaiwannou, Antonis; Lampaki, Sofia; Zaric, Bojan; Branislav, Perin; Dervelegas, Konstantinos; Porpodis, Konstantinos

    2014-01-01

    Pneumothorax occurs when air enters the pleural space. Currently there is increasing incidence of road traffic accidents, increasing awareness of healthcare leading to more advanced diagnostic procedures, and increasing number of admissions in intensive care units are responsible for traumatic (non iatrogenic and iatrogenic) pneumothorax. Pneumothorax has a clinical spectrum from asymptomatic patient to life-threatening situations. Diagnosis is usually made by clinical examination and imaging techniques. In our current work we focus on the treatment of penetrating trauma. PMID:25337403

  20. Oocyte maturation: converting the zebrafish oocyte to the fertilizable egg.

    PubMed

    Lessman, Charles A

    2009-03-01

    The process of oogenesis culminates in steroid-induced oocyte maturation to produce the fertilizable egg. A quintessential biological entity, the egg is central to the production of new individuals. The result of egg fertilization by a sperm cell is the production of the mother of all stem cells (i.e. the zygote). Furthermore, the egg cytoplasm is the only one known to support reprogramming a transplanted nucleus to give rise to an individual (i.e. animal cloning). Zebrafish oocyte maturation is a complex event encompassing a number of cellular changes including germinal vesicle migration (GVM) and dissolution or breakdown (GVD), ooplasmic clearing (OC) with correlated yolk protein changes (YP), development of osmoregulation (OR) in fresh water, the formation of the future embryonic pole, the blastodisc (BF) and activatibility (AC) or cortical maturation. In zebrafish, and many other teleosts, 17alpha, 20beta-dihydroxy-4-pregnen-3-one (17alpha, 20beta-DP) has been shown to be the normal inducer of oocyte maturation. A 17alpha, 20beta-DP membrane-resident receptor mediates oocyte maturation via non-genomic mechanisms that are beginning to be understood. This paper will highlight some of the cellular markers resulting from the signaling initiated by 17alpha, 20beta-DP. By describing these markers, it is hoped that workers in the field will have additional tools to help further elucidate the signaling events of oocyte maturation. PMID:19027744

  1. An overview of oocyte cryopreservation.

    PubMed

    Stachecki, James J; Cohen, Jacques

    2004-08-01

    The ability to cryopreserve human oocytes and store them indefinitely would be beneficial for cancer patients at risk of becoming sterile after therapy, allow women to delay reproduction, and alleviate religious concerns associated with embryo storage. In 1986, Chen was the first to report a pregnancy originating from a frozen-thawed human oocyte. Although over 100 babies have been born from oocyte storage since then, pregnancy rates remain unacceptably low. Adapting embryo cryopreservation techniques to oocyte storage has had limited success and new reproducible methods are needed. Problem areas other than intracellular ice formation and osmotic effects need to be identified. A broad approach of critical analysis should be conducted regarding the entire cryopreservation process from pre-equilibration and cooling, to thawing and stepout. All established facets deserve reanalysis in order to assess which aspects can be optimized or changed so that cellular demise can be avoided and cellular viability enhanced. New methods, including the use of choline-based media and vitrification have proven useful in increasing survival and pregnancy rates in some clinics. Other methods yet untested, such as injection of complex carbohydrates into the oocyte, deserve further studies. Vitrification research has led to the formulation of new ideas and has demonstrated the flexibility of cells to survive cryopreservation. Although successful, vitrification protocols are potentially harmful and technically challenging, due to elevated cryoprotectant concentrations and rapid cooling rates. Bovine embryo vitrification methods have been used to store human oocytes and embryos, particularly blastocysts with some success. Vitrification solutions containing high molecular weight polymers have also proved beneficial by reducing solution toxicity. In general, further advances are needed to improve human oocyte storage before widespread routine clinical use. PMID:15333244

  2. Procaine Induces Cytokinesis in Horse Oocytes via a pH-Dependent Mechanism.

    PubMed

    Leemans, Bart; Gadella, Bart M; Stout, Tom A E; Heras, Sonia; Smits, Katrien; Ferrer-Buitrago, Minerva; Claes, Eline; Heindryckx, Bjrn; De Vos, Winnok H; Nelis, Hilde; Hoogewijs, Maarten; Van Soom, Ann

    2015-07-01

    Coincubating equine gametes in the presence of procaine has been reported to facilitate in vitro fertilization, with cleavage rates exceeding 60%. We report that while procaine does trigger sperm hyperactivation, it independently induces cleavage of equine oocytes. First, we found that procaine (1-5 mM) did not facilitate stallion sperm penetration of equine oocytes but instead induced sperm-independent oocyte cytokinesis in the absence of the second polar body extrusion. Indeed, 56 4% of oocytes cleaved within 2.5 days of exposure to 2.5 mM procaine regardless of sperm presence. However, the cleaved oocytes did not develop beyond 8 to 16 cells, and the daughter cells either lacked nuclei or contained aberrant, condensed DNA fragments. By contrast, intracytoplasmic sperm injection (ICSI) was followed by second polar body extrusion and formation of normal blastocysts. Moreover, neither the calcium oscillations detectable using fura-2 AM staining nor the cortical granule reaction visualized by LCA-FITC staining, after oocyte activation induced by ICSI or ionomycin treatment, were detected after exposing oocytes to 2.5 mM procaine. Instead, procaine initiated an ooplasmic alkalinization, detectable by BCECF-AM staining that was not observed after other treatments. This alkalinization was followed, after an additional 18 h of incubation, by cortical F-actin depolymerization, as demonstrated by reduced actin phalloidin-FITC staining intensity, that resembled preparation for cytokinesis in ICSI-fertilized zygotes. Overall, we conclude that procaine induces cytokinesis in equine oocytes accompanied by aberrant chromatin condensation and division; this explains why embryos produced after exposing equine oocytes to procaine fail to develop beyond the 8- to 16-cell stage. PMID:26085521

  3. Micromanipulation of the human oocyte.

    PubMed

    Nagy, Zsolt Peter

    2003-12-01

    Intracytoplasmic sperm injection (ICSI) provides an excellent outcome in a consistent manner, and is therefore used worldwide as a routine procedure. Since its introduction, few modifications have been made to its methodology. Recently, a combination of ICSI with micro-hole drilling by laser (LA-ICSI) of the zona pellucida appeared to decrease oocyte degeneration rates and to improve embryo quality and implantation. Cytoplasmic transfer is a more recently introduced procedure where the objective is to improve the quality of patients' oocytes by transferring cytoplasm from a good quality donor oocyte, in cases where it is assumed that cytoplasm is compromised. Nuclear transfer, involving exchange of nuclei between donor and receptor oocytes, is still an experimental procedure, the objective being similar to cytoplasmic transfer in improving oocyte/embryo quality. A nuclear transfer procedure involving somatic cells for reproductive purposes should not be used in humans, for ethical and technical considerations. On the other hand, nuclear transfer for therapeutic purposes to obtain stem cells may be considered in respect of its unique potential in medicine. Finally, the most recently emerged new concept under investigation is the haploidization of somatic cells for the purpose of creating artificial gametes. PMID:14748960

  4. Nuclear and Spindle Positioning during Oocyte Meiosis

    PubMed Central

    Fabritius, Amy S.; Ellefson, Marina L.; McNally, Francis J.

    2010-01-01

    Female meiosis is unique in that an asymmetrically positioned meiotic spindle expels chromosomes into tiny, non-developing polar bodies. The extrusion of chromosomes into polar bodies is always mediated by meiotic spindles that are attached to the oocyte cortex by one pole. The asymmetric, cortical positioning of the oocyte meiotic spindle preserves the volume and contents of the oocyte. Recent work in C. elegans and mouse has provided mechanistic details of spindle positioning in oocytes. PMID:20708397

  5. 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

  6. FATE OF INHALED FLY ASH IN HAMSTERS

    EPA Science Inventory

    To determine pulmonary deposition, translocation, and clearance of inhaled fly ash, hamsters received a single 95-min nose-only exposure to neutron-activated fly ash. Over a period of 99 days postexposure, the hamsters were sacrificed in groups of six animals. Lungs, liver, kidne...

  7. 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

  8. 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.

  9. Embryological, clinical and ultrastructural study of human oocytes presenting indented zona pellucida.

    PubMed

    Sousa, M; Teixeira da Silva, J; Silva, J; Cunha, M; Viana, P; Oliveira, E; S, R; Soares, C; Oliveira, C; Barros, A

    2015-02-01

    Human oocyte dysmorphisms attain a large proportion of retrieved oocytes from assisted reproductive technology (ART) treatment cycles. Extracytoplasmic defects involve abnormal morphology of the zona pellucida (ZP), perivitelline space and first polar body. The aim of the present study was to describe a novel dysmorphism affecting the ZP, indented ZP. We also evaluated the clinical, embryological and ultrastructural features of these cases. We evaluated all ART treatment cycles during 7 consecutive years and found 13 treatment cycles (six patients) with all oocytes presenting an indented ZP. In addition, these oocytes presented total or partial absence of the perivitelline space, absence of resistance to ZP and oolemma penetration during microinjection, and low ooplasm viscosity during aspiration. This novel described dysmorphism was recurrent and attained all oocytes in three cases that had more than one treatment cycle. When compared with controls, data showed significant low oocyte maturity (42% versus 81.6%) and high cycle cancellation (30.8% versus 8.5%) rates, normal degeneration (3.4% versus 6.3%) and fertilization rates (69% versus 69.5%), and low pregnancy (15.4% versus 33.3%) and live-birth delivery (7.7% versus 27.7%) rates per cycle. Ultrastructure analysis revealed a zona pellucida structure with large empty electrolucent regions, an outer ZP layer with an indented surface with protuberances and a thick inner ZP that obliterated the perivitelline space. There was evidence of exocytosis of ZP material by the oocyte. In conclusion, oocytes with this novel described dysmorphism (indented ZP) are associated with low maturity, pregnancy and live-birth delivery rates. PMID:23992046

  10. Oocyte control of metabolic cooperativity between oocytes and companion granulosa cells: energy metabolism.

    PubMed

    Sugiura, Koji; Pendola, Frank L; Eppig, John J

    2005-03-01

    Intercellular communication between oocytes and granulosa cells is essential for normal follicular differentiation and oocyte development. Subtraction hybridization was used to identify genes more highly expressed in cumulus cells than in mural granulosa cells of mouse antral follicles. This screen identified six genes involved in glycolysis: Eno1, Pkm2, Tpi, Aldoa, Ldh1, and Pfkp. When oocytes were microsurgically removed from cumulus cell-oocyte complexes, the isolated cumulus cells exhibited decreased expression levels of genes encoding glycolytic enzymes, glycolysis and activity of the tricarboxylic acid (TCA) cycle. These decreases were prevented by culturing the cumulus cells with paracrine factors secreted by fully grown oocytes. Paracrine factors from fully grown oocytes exhibited greater ability than those from growing oocytes to promote expression of genes encoding glycolytic enzymes and glycolysis in the granulosa cells of preantral follicles. However, neither fully grown nor growing oocytes secreted paracrine factors affecting activity of the TCA cycle. These results indicate that oocytes regulate glycolysis and the TCA cycle in granulosa cells in a manner specific to the population of granulosa cells and to the stage of growth and development of the oocyte. Oocytes control glycolysis in granulosa cells by regulating expression levels of genes encoding glycolytic enzymes. Therefore, mouse oocytes control the intercellular metabolic cooperativity between cumulus cells and oocytes needed for energy production by granulosa cells and required for oocyte and follicular development. PMID:15708555

  11. Comparison of in-vitro outcomes from cryopreserved oocytes and sibling fresh oocytes.

    PubMed

    Chamayou, S; Alecci, C; Ragolia, C; Storaci, G; Maglia, E; Russo, E; Guglielmino, A

    2006-06-01

    In Italy, the restrictive IVF law generalizes the indication for oocyte freezing for surplus oocytes in 78.5% of in-vitro assisted reproductive cycles. With a view to understanding better what the prospects for intracytoplasmic sperm injection (ICSI) on frozen-thawed oocytes might be, the consequences of freeze-thaw procedures on fertilization, cleavage rates and embryo quality obtained from frozen-thawed oocytes were studied and compared with the results obtained from sibling fresh oocytes. Eleven IVF and 29 ICSI on 76 and 169 fresh oocytes were performed and the corresponding 40 ICSI on 221 sibling frozen-thawed oocytes. There was no difference in terms of fertilization rate between fresh and sibling frozen-thawed oocytes. The cleavage rate (98.0 and 94.4% with fresh oocytes in IVF and ICSI; 77.3% with frozen-thawed oocytes in ICSI; P < 0.001) and embryo quality (grade I embryos over total embryos: 36.7 and 22.2% with fresh oocytes in IVF and ICSI; 12.1% with frozen-thawed oocytes in ICSI; respectively P < 0.001 and P < 0.05) were statistically lower after oocyte cryopreservation. The significant decrease in meiotic spindle retrieval rate before freezing (62.4%) and after thawing procedures (43.4%; P < 0.001) suggests that cryoconservation induces irreversible damage to microtubule repolymerization. The consequences of oocyte cryopreservation procedures on embryo development are reviewed. PMID:16792849

  12. Spindle Dynamics during Meiosis in Drosophila Oocytes

    PubMed Central

    Endow, Sharyn A.; Komma, Donald J.

    1997-01-01

    Mature oocytes of Drosophila are arrested in metaphase of meiosis I. Upon activation by ovulation or fertilization, oocytes undergo a series of rapid changes that have not been directly visualized previously. We report here the use of the Nonclaret disjunctional (Ncd) microtubule motor protein fused to the green fluorescent protein (GFP) to monitor changes in the meiotic spindle of live oocytes after activation in vitro. Meiotic spindles of metaphase-arrested oocytes are relatively stable, however, meiotic spindles of in vitroactivated oocytes are highly dynamic: the spindles elongate, rotate around their long axis, and undergo an acute pivoting movement to reorient perpendicular to the oocyte surface. Many oocytes spontaneously complete the meiotic divisions, permitting visualization of progression from meiosis I to II. The movements of the spindle after oocyte activation provide new information about the dynamic changes in the spindle that occur upon re-entry into meiosis and completion of the meiotic divisions. Spindles in live oocytes mutant for a lossof-function ncd allele fused to gfp were also imaged. The genesis of spindle defects in the live mutant oocytes provides new insights into the mechanism of Ncd function in the spindle during the meiotic divisions. PMID:9182665

  13. High doses of medroxyprogesterone as the cause of disappearance of adherence of the zona pellucida to an oocyte.

    PubMed

    Jod?owska-Jedrych, Barbara; Jedrych, Marian; Matysiak, W?odzimierz

    2010-10-01

    The zona pellucida (ZP) is an external glycoprotein membrane of oocytes of mammals and embryos in the early stage of their development. ZP first appears in growing ovarian follicles as an extracellular substance between the oocyte and granular cells. The zona pellucid markedly affects the development and maturation of the oocyte. The morphology of the ZP-oocyte complex allows a more precise determination of the oocyte maturity. According to numerous experimental studies, ZP is essential for preimplantation embryonic development of humans and other mammals. It prevents dispersion of blastomeres and enhances their mutual interactions. ZP is a dynamic structure responsible for the provision of nutrients to early forms of oocytes in mammals. The aim of the present study was untrastructural evaluation of the ZP-oocyte contact during inhibited ovulation. Female white rats (Wistar strain) received a suspension of medroxyprogesterone acetate (MPA) in incremental intramuscular bolus doses of 3.7 mg (therapeutic dose), 7.4 mg and 11.1 mg. The animals were decapitated 5 days after the administration of MPA. Ovarian sections were evaluated under a transmission electron microscope (TEM) Zeiss EM 900. Morphometric analysis of ZP was conducted using the cell imaging system by Olympus. In females exposed to therapeutic doses of MPA, ZP showed the structure of granular-fibrous reticulum of a medium electron density with single cytoplasmic processes originating from the surrounding structures. The oocyte cell membrane generated single, delicate processes directed toward ZP. Microvilli of the oocyte were short and thin. In the group receiving 7.4 mg of MPA, ZP had the structure of a delicate, loose granular-fibrous reticulum, and the oocyte cell membrane generated single microvilli directed toward ZP. In both those groups, the close ZP-oocyte contact was observed. Otherwise, in the group exposed to the highest MPA doses (11.1 mg), thicker and more numerous oocyte microvilli were found, which did not penetrate ZP matrix. They were dense, irregularly separated contour, forming a barrier between ZP and oocyte. The present findings are likely to suggest that MPA has inhibiting effects on the synthesis of binding proteins and causes the loss of the oocyte contact with ZP. PMID:20714762

  14. How is plasminogen/plasmin system contributing to regulate sperm entry into the oocyte?

    PubMed

    Grulln, Luis A; Gadea, Joaqun; Mondjar, Irene; Mats, Carmen; Romar, Raquel; Coy, Pilar

    2013-09-01

    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. 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.; Garca, 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-thawed spermatozoa, producing live pups. The ability to cryopreserve mouse gametes for LAIVF may facilitate management of large-scale transgenic mouse production facilities. PMID:24618785

  16. 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.

  17. 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.

  18. 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.

  19. Apoptosis in mammalian oocytes: a review.

    PubMed

    Tiwari, Meenakshi; Prasad, Shilpa; Tripathi, Anima; Pandey, Ashutosh N; Ali, Irfan; Singh, Arvind K; Shrivastav, Tulsidas G; Chaube, Shail K

    2015-08-01

    Apoptosis causes elimination of more than 99% of germ cells from cohort of ovary through follicular atresia. Less than 1% of germ cells, which are culminated in oocytes further undergo apoptosis during last phases of oogenesis and depletes ovarian reserve in most of the mammalian species including human. There are several players that induce apoptosis directly or indirectly in oocytes at various stages of meiotic cell cycle. Premature removal of encircling granulosa cells from immature oocytes, reduced levels of adenosine 3',5'-cyclic monophosphate and guanosine 3',5'-cyclic monophosphate, increased levels of calcium (Ca(2+)) and oxidants, sustained reduced level of maturation promoting factor, depletion of survival factors, nutrients and cell cycle proteins, reduced meiotic competency, increased levels of proapoptotic as well as apoptotic factors lead to oocyte apoptosis. The BH3-only proteins also act as key regulators of apoptosis in oocyte within the ovary. Both intrinsic (mitochondria-mediated) as well as extrinsic (cell surface death receptor-mediated) pathways are involved in oocyte apoptosis. BID, a BH3-only protein act as a bridge between both apoptotic pathways and its cleavage activates cell death machinery of both the pathways inside the follicular microenvironment. Oocyte apoptosis leads to the depletion of ovarian reserve that directly affects reproductive outcome of various mammals including human. In this review article, we highlight some of the important players and describe the pathways involved during oocyte apoptosis in mammals. PMID:25958165

  20. Calcium waves occur as Drosophila oocytes activate

    PubMed Central

    Kaneuchi, Taro; Sartain, Caroline V.; Takeo, Satomi; Horner, Vanessa L.; Buehner, Norene A.; Aigaki, Toshiro; Wolfner, Mariana F.

    2015-01-01

    Egg activation is the process by which a mature oocyte becomes capable of supporting embryo development. In vertebrates and echinoderms, activation is induced by fertilization. Molecules introduced into the egg by the sperm trigger progressive release of intracellular calcium stores in the oocyte. Calcium wave(s) spread through the oocyte and induce completion of meiosis, new macromolecular synthesis, and modification of the vitelline envelope to prevent polyspermy. However, arthropod eggs activate without fertilization: in the insects examined, eggs activate as they move through the female’s reproductive tract. Here, we show that a calcium wave is, nevertheless, characteristic of egg activation in Drosophila. This calcium rise requires influx of calcium from the external environment and is induced as the egg is ovulated. Pressure on the oocyte (or swelling by the oocyte) can induce a calcium rise through the action of mechanosensitive ion channels. Visualization of calcium fluxes in activating eggs in oviducts shows a wave of increased calcium initiating at one or both oocyte poles and spreading across the oocyte. In vitro, waves also spread inward from oocyte pole(s). Wave propagation requires the IP3 system. Thus, although a fertilizing sperm is not necessary for egg activation in Drosophila, the characteristic of increased cytosolic calcium levels spreading through the egg is conserved. Because many downstream signaling effectors are conserved in Drosophila, this system offers the unique perspective of egg activation events due solely to maternal components. PMID:25564670

  1. Intracytoplasmic sperm injection in dysmorphic human oocytes.

    PubMed

    Alikani, M; Palermo, G; Adler, A; Bertoli, M; Blake, M; Cohen, J

    1995-11-01

    Fertilisation and development of dysmorphic human oocytes recovered from hyperstimulated ovaries have been evaluated following intracytoplasmic sperm injection (ICSI) for treatment of male infertility. A total of 2968 oocytes at metaphase II of meiosis were injected, of which 806 (27.2%) were dysmorphic at the light microscopic level. Cytoplasmic abnormalities included granularity, areas of necrosis, organelle clustering, vacuoles, and accumulating saccules of smooth endoplasmic reticulum. Anomalies of the first polar body and zona pellucida, as well as non-spherical shapes of oocytes, were also noted. Contrary to previous findings linking some dysmorphisms to non-assisted fertilisation failure, in this study no single abnormality led to a reduction in the fertilisation rate, nor was fertilisation compromised in oocytes with multiple abnormalities. The incidence of normal fertilisation (two pronuclei and two polar bodies) was 69% in both the dysmorphic and non-dysmorphic oocytes. While overall pregnancy and implantation results were not altered in the group of patients (n = 242) in whom at least one dysmorphic oocyte was injected, exclusive replacement of embryos which originated from dysmorphic oocytes led to a higher incidence of early pregnancy loss. It is concluded that aberrations in the morphology of human oocytes--most probably a product of controlled ovarian stimulation--are of little or no consequence to fertilisation or early cleavage after ICSI. It is possible, however, that these embryos have a reduced potential for implantation and further development. PMID:8730892

  2. Calcium waves occur as Drosophila oocytes activate.

    PubMed

    Kaneuchi, Taro; Sartain, Caroline V; Takeo, Satomi; Horner, Vanessa L; Buehner, Norene A; Aigaki, Toshiro; Wolfner, Mariana F

    2015-01-20

    Egg activation is the process by which a mature oocyte becomes capable of supporting embryo development. In vertebrates and echinoderms, activation is induced by fertilization. Molecules introduced into the egg by the sperm trigger progressive release of intracellular calcium stores in the oocyte. Calcium wave(s) spread through the oocyte and induce completion of meiosis, new macromolecular synthesis, and modification of the vitelline envelope to prevent polyspermy. However, arthropod eggs activate without fertilization: in the insects examined, eggs activate as they move through the female's reproductive tract. Here, we show that a calcium wave is, nevertheless, characteristic of egg activation in Drosophila. This calcium rise requires influx of calcium from the external environment and is induced as the egg is ovulated. Pressure on the oocyte (or swelling by the oocyte) can induce a calcium rise through the action of mechanosensitive ion channels. Visualization of calcium fluxes in activating eggs in oviducts shows a wave of increased calcium initiating at one or both oocyte poles and spreading across the oocyte. In vitro, waves also spread inward from oocyte pole(s). Wave propagation requires the IP3 system. Thus, although a fertilizing sperm is not necessary for egg activation in Drosophila, the characteristic of increased cytosolic calcium levels spreading through the egg is conserved. Because many downstream signaling effectors are conserved in Drosophila, this system offers the unique perspective of egg activation events due solely to maternal components. PMID:25564670

  3. The equine oocyte: factors affecting meiotic and developmental competence.

    PubMed

    Hinrichs, Katrin

    2010-08-01

    There is currently much interest in assisted reproduction techniques in the horse, however, many aspects of oocyte maturation, fertilization, and embryo development in the horse differ from those in other species. Because of the close attachment of the equine oocyte to the follicle wall, scraping of the follicle is the most effective method for oocyte recovery. A notable feature of equine oocytes is that those with expanded cumuli (Ex oocytes), which originate from atretic follicles, have higher meiotic competence (ability to mature to metaphase II in vitro) than do oocytes with compact cumuli (Cp oocytes). Cp oocytes originate in viable follicles but are largely juvenile. Recovery and culture of equine oocytes immediately after slaughter yields a higher maturation rate than that obtained from oocytes after ovary storage; this is related to damage to chromatin in Cp oocytes during storage. In contrast, developmental competence (rate of blastocyst development in vitro) is higher in oocytes recovered from the ovary after a delay. The optimum duration of maturation varies based on cumulus morphology and time of recovery from the ovary, but there is no difference in developmental competence between Ex and Cp oocytes. Because standard in vitro fertilization is not repeatable in the horse, oocyte transfer (surgical transfer of oocytes to the oviducts of inseminated mares) has been developed to allow fertilization of isolated oocytes. Fertilization in vitro may be achieved using intracytoplasmic sperm injection; culture of injected oocytes in a medium with high glucose can yield over 30% blastocyst development. PMID:20652997

  4. Control of oocyte meiotic maturation and fertilization.

    PubMed Central

    Greenstein, David

    2005-01-01

    Sexual reproduction depends upon meiosis for the generation of haploid gamete nuclei, which unite after fertilization to form the diploid zygote. The oocytes of most animal species arrest during meiotic prophase, and complete meiosis in response to intercellular signaling in a process called meiotic maturation. Oocyte meiotic maturation is defined by the transition between diakinesis and metaphase of meiosis I and is accompanied by nuclear envelope breakdown, rearrangement of the cortical cytoskeleton, and meiotic spindle assembly. Thus, the meiotic maturation process is essential for meiosis and prepares the oocyte for fertilization. In C. elegans, the processes of meiotic maturation, ovulation, and fertilization are temporally coupled: sperm utilize the major sperm protein as a hormone to trigger oocyte meiotic maturation, and in turn, the maturing oocyte signals its own ovulation thereby facilitating fertilization. This chapter highlights recent advances in understanding meiotic maturation signaling and gametic interactions required for fertilization. PMID:18050412

  5. 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

  6. [FERTILITY PRESERVATION OF WOMEN: OOCYTE VITRIFICATION].

    PubMed

    Montserrat, Pallas Seijas

    2015-09-01

    Cryopreservation ofhuman oocytes to delay fertility also be an option for women who are going to be subjected to a cancer/autoimmune treatment. It allows for creating a bank of oocytes for donation in assisted reproduction centers. The legislation allows the use of cryopreserved oocytes throughout the reproductive life of women with what conservation could last up to 48-50 years. Oocyte vitrification is a ultrafast freezing method in which cryoprotectants are used to prevent the formation of ice crystals within the cell. Treatment for oocyte vitrification process is similar to IVF treatment, ending at the time of obtaining the ova. The eggs obtained in the laboratory are classified according to maturity and quality. The apartments will be cryopreserved by vitrification technique tanks and maintained in liquid nitrogen until used for reproductive purposes. PMID:26738231

  7. Developmental stages of primary oocytes in turkeys.

    PubMed

    Carlson, J L; Bakst, M R; Ottinger, M A

    1996-12-01

    Little is known about the growth and differentiation of the primary oocyte in the sexually mature chicken or turkey hen. In this study, primary oocytes from turkey hens in egg production were examined by light and electron microscopy. Based on oocyte and germinal vesicle (GV) diameters and organelle morphology and distribution, the sequential development of the primary oocyte was divided into five stages. No Balbiani body was observed in Stage I oocytes (< 80 microns in diameter). Pleomorphic mitochondria were localized around the GV and multivesicular bodies were scattered in the ooplasm. By Stage II (81 to 150 microns), the Balbiani body was observed adjacent to the GV. Pleomorphic mitochondria, macrobodies, and smooth endoplasmic reticulum (SER) were associated with the Balbiani body. Lipid droplets were predominantly localized to the periphery of the oocyte. The Balbiani body was partially dispersed by Stage III (151 to 350 microns) and associated organelles appeared in clusters in the ooplasm. Golgi and SER were observed immediately subjacent to the oolemma. Stage IV oocytes (351 to 500 microns) were characterized by the absence of the Balbiani body, a more centrally located GV, and the redistribution of the mitochondria to the periphery of the oocyte. Throughout the ooplasm was vesicular SER. By Stage V (501 to 800 microns), zonation of the organelles was completed with the mitochondrial ring immediately subjacent to the oolemma and a concentric layer of lipid droplets subjacent to the mitochondrial ring. The GV was in the periphery of the oocyte. Organelle and inclusion redistribution and organelle pleomorphism were presumed to be reflective of increasing metabolic and transport requirements of the growing oocyte in the mature turkey hen. PMID:9000285

  8. 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

  9. Maternal gene transcription in mouse oocytes: genes implicated in oocyte maturation and fertilization.

    PubMed

    Cui, Xiang-Shun; Li, Xing-Yu; Yin, Xi-Jun; Kong, Il Keun; Kang, Jason-Jongho; Kim, Nam-Hyung

    2007-04-01

    Maternal gene expression is an important biological process in oocyte maturation and early cleavage. To gain insights into oocyte maturation and early embryo development, we used microarray analysis to compare the gene expression profiles of germinal vesicle (GV)- and metaphase II (MII)-stage oocytes. The differences in spot intensities were normalized and grouped using the Avadis Prophetic software platform. Of the 12164 genes examined, we found 1682 genes with more highly expression in GV-stage oocytes than in MII-stage oocytes, while 1936 genes were more highly expressed in MII-stage oocytes (P<0.05). The genes were grouped on the basis of the Panther classification system according to their involvement in particular biological processes. The genes that were up-regulated in GV oocytes were more likely to be involved in protein metabolism and modification, the mitotic cell cycle, electron transport, or fertilization or belong to the microtubule/cytoskeletal protein family. The genes specifically upregulated in the MII oocytes were more likely to be involved in DNA replication, amino acid metabolism, or expression of G protein-coupled receptors and signaling molecules. Identification of genes that are preferentially expressed at particular oocyte maturation stages provides insights into the complex gene regulatory networks that drive oocyte maturation and fertilization. PMID:17179655

  10. Noninvasive bovine oocyte quality assessment: possibilities of a single oocyte culture.

    PubMed

    Goovaerts, I G F; Leroy, J L M R; Jorssen, E P A; Bols, P E J

    2010-12-01

    Although bovine embryos are routinely produced in vitro for several decades, there still exists a critical need for techniques to accurately predict the oocyte's developmental competence in a noninvasive way, before the in vitro embryo production procedure. In this review, several noninvasive methods to evaluate oocyte quality are discussed, such as morphological assessment of the cumulus oocyte complex and the use of brilliant cresyl blue. Because an individual oocyte and embryo culture method can possibly generate additional insights into the factors that determine oocyte quality, the second part of this review summarizes the state of the art of bovine single oocyte culture. The optimization of individual in vitro embryo production can obviously accelerate the quest for better noninvasive oocyte quality markers, because more information about the oocyte's requirements and intrinsic quality will be revealed. Although each step of in vitro culture has to be re-examined in light of the hampered production of single embryos, the reward at the end will be substantial. Individual scored oocytes will be traceable along the in vitro embryo production procedure and the final blastocyst outcome can be linked to the original oocyte quality and follicular environment without the bias caused by simultaneously developing embryos. PMID:20708251

  11. Diffused Intra-Oocyte Hydrogen Peroxide Activates Myeloperoxidase and Deteriorates Oocyte Quality.

    PubMed

    Khan, Sana N; Shaeib, Faten; Najafi, Tohid; Kavdia, Mahendra; Gonik, Bernard; Saed, Ghassan M; Goud, Pravin T; Abu-Soud, Husam M

    2015-01-01

    Hydrogen peroxide (H2O2) is a relatively long-lived signaling molecule that plays an essential role in oocyte maturation, implantation, as well as early embryonic development. Exposure to relatively high levels of H2O2 functions efficiently to accelerate oocyte aging and deteriorate oocyte quality. However, little precise information exists regarding intra-oocyte H2O2 concentrations, and its diffusion to the oocyte milieu. In this work, we utilized an L-shaped amperometric integrated H2O2-selective probe to directly and quantitatively measure the real-time intra-oocyte H2O2 concentration. This investigation provides an exact measurement of H2O2 in situ by reducing the possible loss of H2O2 caused by diffusion or reactivity with other biological systems. This experiment suggests that the intra-oocyte H2O2 levels of oocytes obtained from young animals are reasonably high and remained constant during the procedure measurements. However, the intra-oocyte H2O2 concentration dropped significantly (40-50% reduction) in response to catalase pre-incubation, suggesting that the measurements are truly H2O2 based. To further confirm the extracellular diffusion of H2O2, oocytes were incubated with myeloperoxidase (MPO), and the diffused H2O2 triggered MPO chlorinating activity. Our results show that the generated hypochlorous acid (HOCl) facilitated the deterioration in oocyte quality, a process that could be prevented by pre-incubating the oocytes with melatonin, which was experimentally proven to be oxidized utilizing HPLC methods. This study is the first to demonstrate direct quantitative measurement of intracellular H2O2, and its extracellular diffusion and activation of MPO as well as its impact on oocyte quality. These results may help in designing more accurate treatment plans in assisted reproduction under inflammatory conditions. PMID:26197395

  12. Diffused Intra-Oocyte Hydrogen Peroxide Activates Myeloperoxidase and Deteriorates Oocyte Quality

    PubMed Central

    Khan, Sana N.; Shaeib, Faten; Najafi, Tohid; Kavdia, Mahendra; Gonik, Bernard; Saed, Ghassan M.; Goud, Pravin T.; Abu-Soud, Husam M.

    2015-01-01

    Hydrogen peroxide (H2O2) is a relatively long-lived signaling molecule that plays an essential role in oocyte maturation, implantation, as well as early embryonic development. Exposure to relatively high levels of H2O2 functions efficiently to accelerate oocyte aging and deteriorate oocyte quality. However, little precise information exists regarding intra-oocyte H2O2 concentrations, and its diffusion to the oocyte milieu. In this work, we utilized an L-shaped amperometric integrated H2O2-selective probe to directly and quantitatively measure the real-time intra-oocyte H2O2 concentration. This investigation provides an exact measurement of H2O2 in situ by reducing the possible loss of H2O2 caused by diffusion or reactivity with other biological systems. This experiment suggests that the intra-oocyte H2O2 levels of oocytes obtained from young animals are reasonably high and remained constant during the procedure measurements. However, the intra-oocyte H2O2 concentration dropped significantly (40-50% reduction) in response to catalase pre-incubation, suggesting that the measurements are truly H2O2 based. To further confirm the extracellular diffusion of H2O2, oocytes were incubated with myeloperoxidase (MPO), and the diffused H2O2 triggered MPO chlorinating activity. Our results show that the generated hypochlorous acid (HOCl) facilitated the deterioration in oocyte quality, a process that could be prevented by pre-incubating the oocytes with melatonin, which was experimentally proven to be oxidized utilizing HPLC methods. This study is the first to demonstrate direct quantitative measurement of intracellular H2O2, and its extracellular diffusion and activation of MPO as well as its impact on oocyte quality. These results may help in designing more accurate treatment plans in assisted reproduction under inflammatory conditions. PMID:26197395

  13. A cytochemical analysis of the follicular cells and the yolk in the growing oocytes of Octopus vulgaris (Cephalopoda, Mollusca).

    PubMed

    Bolognari, A; Carmignani, M P; Zaccone, G

    1976-01-01

    From an examination of the structural and cytochemical data obtained on the follicular epithelium and on the growing oocytes of Octopus vulgaris it has been possible to establish that, during the evitellogenetic period, the follicular cells penetrate into the oocyte cytoplasm and assume the form of cords. The yolk, which meanwhile has been constituted also through the probable contribution of material metiated by the follicular cells, is seen to be rich in neutral glycoproteins, proteins with sulphydrilic and thiolic radicals and proteins tyrosine and tryptophan containing, but is lacking in glycogen and in acid mucopolysaccharides. PMID:135466

  14. 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.

  15. Cytoplasmic polyadenylation in mammalian oocyte maturation.

    PubMed

    Reyes, Juan M; Ross, Pablo J

    2016-01-01

    Oocyte developmental competence is the ability of the mature oocyte to be fertilized and subsequently drive early embryo development. Developmental competence is acquired by completion of oocyte maturation, a process that includes nuclear (meiotic) and cytoplasmic (molecular) changes. Given that maturing oocytes are transcriptionally quiescent (as are early embryos), they depend on post-transcriptional regulation of stored transcripts for protein synthesis, which is largely mediated by translational repression and deadenylation of transcripts within the cytoplasm, followed by recruitment of specific transcripts in a spatiotemporal manner for translation during oocyte maturation and early development. Motifs within the 3' untranslated region (UTR) of messenger RNA (mRNA) are thought to mediate repression and downstream activation by their association with binding partners that form dynamic protein complexes that elicit differing effects on translation depending on cell stage and interacting proteins. The cytoplasmic polyadenylation (CP) element, Pumilio binding element, and hexanucleotide polyadenylation signal are among the best understood motifs involved in CP, and translational regulation of stored transcripts as their binding partners have been relatively well-characterized. Knowledge of CP in mammalian oocytes is discussed as well as novel approaches that can be used to enhance our understanding of the functional and contributing features to transcript CP and translational regulation during mammalian oocyte maturation. WIREs RNA 2016, 7:71-89. doi: 10.1002/wrna.1316 For further resources related to this article, please visit the WIREs website. PMID:26596258

  16. Oocyte ageing and its cellular basis.

    PubMed

    Eichenlaub-Ritter, Ursula

    2012-01-01

    Aneuploidy is extremely high in aged human oocytes. Its cellular origin has been elusive. Trisomy data implicate predominantly meiosis I errors in the genesis of oocyte aneuploidy. Susceptible recombination patterns increase risks for nondisjunction. Cytogenetic analyses of aged human oocytes and embryos from assisted reproduction (ART) suggest that aneuploidy primarily relates to precocious chromatid separation. Oocytes express a spindle assembly checkpoint (SAC), but do not arrest maturation in the presence of improperly attached or single, unattached chromosomes. The SAC may be more permissive by altered gene expression in aged oocytes. Aged oocytes frequently exhibit precocious loss of chromosome cohesion. In experimental models, cohesion cannot be restored once lost, a process possibly occurring during long meiotic arrest. Maternal age, hormonal stimulation, disturbed metabolism, and depletion of the follicle pool contribute to mitochondrial dysfunction, spindle aberrations, and errors in chromosome segregation. Caloric restriction and antioxidants reduce mitochondrial dysfunction and aneuploidy in aged rodents' oocytes. Loss of chromosome cohesion appears to be a major risk factor for aneuploidy by disturbing the sequential separation of homologs and chromatids. A permissive SAC, the presence of risky meiotic exchanges, changes in expression, and failures to resolve improper chromosome attachments, as well as mitochondrial dysfunction may synergistically increase susceptibility to meiotic errors. A healthy life style, mild stimulation and an optimal environment may delay ageing and sustain control over chromosome disjunction, whereas loss of cohesion appears to be irreversible. PMID:23417406

  17. Biochemical studies of isolated hamster tracheal epithelium.

    PubMed Central

    Kaufman, D G

    1976-01-01

    The epithelial lining of respiratory air passageways is a primary target tissue for toxicity and carcinogenesis in man and in animal models of human disease. The importance of this target tissue was the basis for development of methods to study its biochemistry, and with this information to distinguish the unique properties of this tissue from properties common to all cell types. Biochemical methods employed labeling of macromolecules in isolated hamster treacheas during brief (less than 4 hr) incubation in vitro. Studies of RNA metabolism in isolated tracheas demonstrated a pattern of maturation of ribosomal RNA like that shown for other cell types. Alterations in RNA metabolism were observed in isolated tracheas obtained from vitamin A-deficient hamsters and hamsters previously treated by intratracheal administration of benzo[a]pyrene (BP) plus ferric oxide (Fe2O3) in vivo. Studies with toyocamycin, actinomycin D, and alpha-amanitin, all inhibitors of RNA metabolism, were performed to characterize the class of RNA molecules with a decreased proportion of labeling in tracheas from vitamin A deficient hamsters. In another series of experiments, BP was shown to bind to DNA in epithelial cells of isolated tracheas. The quantity of BP binding was increased by prior intratracheal treatment of hamsters with BP plus Fe2O3 in vivo, this induced binding was inhibited by addition of 7,8-benzoflavone to the incubation medium. Increased BP binding was also observed in isolated tracheas from hamsters believed to be in states of increased susceptibility to respiratory carcinogenesis in vivo. The results show that biochemical studies are feasible with this tissue. Furthermore, a number of questions of importance with regard to this target epithelium are best studied directly in its constituent cells. PMID:1017424

  18. Comparison of activation ability between feline and bovine oocytes.

    PubMed

    Tanihara, Fuminori; Kaedei, Yukine; Namula, Zhao; Luu, Vien Viet; Sato, Yoko; Wittayarat, Manita; Taniguchi, Masayasu; Otoi, Takeshige

    2013-12-01

    Research comparing the activation sensitivity of oocytes to chemical treatment among mammalian species remains limited. We compared the activation ability of oocytes from bovine and feline ovaries when treated with ethanol alone, with ethanol and cycloheximide, and without any chemical treatment; the oocytes were then cultured for 72 h. After in vitro maturation (IVM), 5% of feline oocytes were activated and 1% were cleaved, whereas there were no prematurely activated bovine oocytes. Activation rates with ethanol and ethanol/cycloheximide were significantly higher (P < 0.01) in bovine oocytes than in feline oocytes (74.2% vs. 34.1% and 86.3% vs. 52.5%, respectively). Thus, our findings indicate that feline oocytes can be prematurely activated by the end of IVM, and that bovine oocytes may have a higher sensitivity of parthenogenetic activation to chemical treatment than do feline oocytes. PMID:23974933

  19. Penetration of concrete targets

    SciTech Connect

    Forrestal, M.J.; Cargile, J.D.; Tzou, R.D.Y.

    1993-08-01

    We developed penetration equations for ogive-nosed projectiles that penetrated concrete targets after normal impact. Our penetration equations predict axial force on the projectile nose, rigid-body motion, and final penetration depth. For target constitutive models, we conducted triaxial material experiments to confining pressures of 600 MPa and curve-fit these data with a linear pressure-volumetric strain relation and with a linear Mohr-Coulomb, shear strength-pressure relation. To verify our penetration equations, we conducted eleven penetration experiments with 0.90 kg, 26.9-mm-diameter, ogive-nosed projectiles into 1.37-m-diameter concrete targets with unconfined compressive strengths between 32-40 MPa. Predictions from our penetration equation are compared with final penetration depth measurements for striking velocities between 280--800 m/s.

  20. Acute infection of encephalomyocarditis (EMC) virus in Syrian hamsters.

    PubMed

    Sugawara, Y; Hirasawa, K; Takeda, M; Han, J S; Doi, K

    1991-06-01

    One-month-old Syrian hamsters of the APA and Std: golden strains were inoculated intraperitoneally with 10(5) PFU/head of the D variant of encephalomyocarditis (EMC) virus and examined virologically and pathologically up to 7 days after inoculation. APA hamsters developed apparent hyperglycemia due to pancreatic islet cell damage while Std:golden hamsters did not. Hamsters of both strains showed clear histopathologic changes in the testis with prominent viral replication as well as in the brain, heart and exocrine pancreas. The susceptibility to EMC virus-infection was higher in males than in females and in APA than in Std: golden hamsters. PMID:1653039

  1. Maternal factors required for oocyte developmental competence in mice

    PubMed Central

    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

    2013-01-01

    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. PMID:23673344

  2. Oocyte environment: follicular fluid and cumulus cells are critical for oocyte health.

    PubMed

    Dumesic, Daniel A; Meldrum, David R; Katz-Jaffe, Mandy G; Krisher, Rebecca L; Schoolcraft, William B

    2015-02-01

    Bidirectional somatic cell-oocyte signaling is essential to create a changing intrafollicular microenvironment that controls primordial follicle growth into a cohort of growing follicles, from which one antral follicle is selected to ovulate a healthy oocyte. Such intercellular communications allow the oocyte to determine its own fate by influencing the intrafollicular microenvironment, which in turn provides the necessary cellular functions for oocyte developmental competence, which is defined as the ability of the oocyte to complete meiosis and undergo fertilization, embryogenesis, and term development. These coordinated somatic cell-oocyte interactions attempt to balance cellular metabolism with energy requirements during folliculogenesis, including changing energy utilization during meiotic resumption. If these cellular mechanisms are perturbed by metabolic disease and/or maternal aging, molecular damage of the oocyte can alter macromolecules, induce mitochondrial mutations, and reduce adenosine triphosphate production, all of which can harm the oocyte. Recent technologies are now exploring transcriptional, translational, and post-translational events within the human follicle with the goal of identifying biomarkers that reliably predict oocyte quality in the clinical setting. PMID:25497448

  3. 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. PMID:22658324

  4. Quantitative Microinjection of Mouse Oocytes and Eggs

    NASA Astrophysics Data System (ADS)

    Kline, Douglas

    Quantitative microinjection is used to introduce known quantities of molecules or probes into single cells to examine cellular function. The relatively large mammalian oocyte or egg is easily manipulated and can be injected with impermeant reagents including a variety of signaling molecules and fluorescent probes. Techniques have been developed to inject picoliter quantities of solution into oocytes and eggs with precision and reliability. The methods described here outline the quantitative injection procedures as they are used to inject mouse oocytes and eggs in a culture dish on the stage on an inverted microscope. The techniques are applicable to the oocytes, eggs, and early embryos of most mammalian species. Included are some general instructions on fabrication of transfer pipettes, holding pipettes, beveled injection pipettes, and equipment for quantitative injection.

  5. Oocyte Meiotic Spindle Assembly and Function.

    PubMed

    Severson, Aaron F; von Dassow, George; Bowerman, Bruce

    2016-01-01

    Gametogenesis in animal oocytes reduces the diploid genome content of germline precursors to a haploid state in gametes by discarding ¾ of the duplicated chromosomes through a sequence of two meiotic cell divisions called meiosis I and II. The assembly of the microtubule-based spindle structure that mediates this reduction in genome content remains poorly understood compared to our knowledge of mitotic spindle assembly and function. In this review, we consider the diversity of oocyte meiotic spindle assembly and structure across animal phylogeny, review recent advances in our understanding of how animal oocytes assemble spindles in the absence of the centriole-based microtubule-organizing centers that dominate mitotic spindle assembly, and discuss different models for how chromosomes are captured and moved to achieve chromosome segregation during oocyte meiotic cell division. PMID:26970614

  6. Exploring RNA virus replication in Xenopus oocytes.

    PubMed

    Gamarnik, Andrea V; Andino, Raul

    2006-01-01

    Microinjection of poliovirus RNA in Xenopus oocytes initiates a complete and authentic viral replication cycle that yields newly synthesized infectious virus. This system can be used to study the molecular mechanism of the different steps involved in virus replication. Interestingly, viral replication only occurs if poliovirus RNA is coinjected with factors present in HeLa extracts. We have determined that two HeLa cell factors are required for viral replication in oocytes, one involved in initiation of translation (polio translation factor) and the other in RNA synthesis. Thus, microinjection in oocytes provides a strategy to identify and further analyze the function of these host cell factors and to biochemically dissect the mechanism of initiation of poliovirus translation and RNA synthesis. Here, we review protocols, approaches, and potential issues that can be addressed using the oocyte system. PMID:16739737

  7. Proteomic profiling of murine oocyte maturation.

    PubMed

    Vitale, Alejandra M; Calvert, Meredith E Kennedy; Mallavarapu, Mallika; Yurttas, Piraye; Perlin, Julie; Herr, John; Coonrod, Scott

    2007-05-01

    In an effort to better understand oocyte function, we utilized two-dimensional (2D) electrophoresis and mass spectrometry to identify proteins that are differentially expressed during murine oocyte maturation. Proteins from 500 germinal vesicle (GV) and metaphase II-(MII) arrested oocytes were extracted, resolved on 2D electrophoretic gels, and stained with silver. Analysis of the gels indicated that 12 proteins appeared to be differentially expressed between the GV and MII stage. These proteins were then cored from the 2D gels and identified by mass spectrometry as: transforming acidic coiled-coil protein 3 (TACC3), heat shock protein 105 (HSP105), programmed cell death six-interacting protein (PDCD6IP), stress-inducible phosphoprotein (STI1), importin alpha2, adenylsuccinate synthase (ADDS), nudix, spindlin, lipocalin, lysozyme, translationally controlled tumor protein (TCTP), and nucleoplasmin 2 (NPM2). Interestingly, PDCD6IP, importin alpha2, spindlin, and NPM2 appear slightly larger in mass and more acidic on the MII oocyte gel compared to the GV oocyte gel, suggesting that they may be post-translationally modified during oocyte maturation. Given NPM2 is an oocyte-restricted protein, we chose to further investigate its properties during oocyte maturation and preimplantation development. Real-Time RT-PCR showed that NPM2 mRNA levels rapidly decline at fertilization. Indirect immunofluorescence analysis showed that, with the exception of cortical localization in MII-arrested oocytes, NPM2 is localized to the nucleus of both GV stage oocytes and all stages of preimplantation embryos. We then performed one-dimensional (1D) western blot analysis of mouse oocytes and preimplantation embryos and found that, as implicated by the 2D gel comparison, NPM2 undergoes a phosphatase-sensitive electrophoretic mobility shift during the GV to MII transition. The slower migrating NPM2 form is also present in pronuclear embryos but by the two-cell stage, the majority of NPM2 exists as the faster migrating form, which persists to the blastocyst stage. PMID:17044029

  8. 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

  9. IN VITRO CULTURE OF POSTIMPLANTATION HAMSTER EMBRYOS

    EPA Science Inventory

    In vitro culture of intact rat and mouse embryos has been described extensively, but information on the culture of other species is sparse. The present study examined some culture requirements of early somite stage hamster embryos and assessed the embryotoxic effects of sodium sa...

  10. 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

  11. 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.

  12. 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.

  13. The oocyte of a new world marsupial, Monodelphis domestica: structure, formation, and function of the enveloping mucoid layer.

    PubMed

    Phillips, D M; Fadem, B H

    1987-06-01

    Ovulated oocytes of the gray short-tailed opossum Monodelphis domestica are surrounded by a thin zona pellucida and are devoid of a cumulus oophorus. In the ampulla of the oviduct, oocytes acquire a thick mucoid layer composed of concentrically arranged fibrillar material. Exocytosis by the secretory cells of the oviductal epithelium occurs in the region of the oviduct adjacent to the egg. This suggests that the oocyte-zona-mucus layer complex may influence the oviductal epithelium to secrete. During secretion, fibrillar contents of the secretion granules appear to be transformed into membranous material which presumably becomes fibrillar again as it is incorporated into the forming mucoid layer. Spermatozoa (which are known to pair in the cauda epididymis) are found in pairs and with intact acrosomes in the mucoid layer of fertilized eggs. This suggests that spermatozoa of Mondelphis remain paired until they reach the zona pellucida and that the acrosome functions in zona binding and/or penetration. PMID:3612053

  14. Involvement of steroid hormones on in vitro maturation of pig oocytes.

    PubMed

    Dode, Margot Alves Nunes; Graves, Charles

    2002-01-15

    The purpose of this study was to determine if the addition of steroid hormones into the culture medium could influence the in vitro maturation of pig oocytes. The cumulus-oocyte complexes (COCs). collected from follicles of 2-5 mm diameter, were matured in steroid-free medium supplemented with various concentrations of estradiol-17beta (0-3000 ng/ml), progesterone (0-5000 ng/ml) and testosterone (0-300 ng/ml). The COCs were cultured for 42 h, then fertilized in vitro. We analyzed nuclear and cytoplasmic maturation with lacmoid stain 20 h after in vitro insemination. We observed no significant effect (P > 0.05) on the percentage of oocytes completing nuclear or cytoplasmic maturation or the number of sperm penetrating each oocyte for any concentration of progesterone, estradiol-17beta or testosterone. Similarly, adding a combination of those hormones to the medium did not significantly (P > 0.05) affect any of the criteria. In order to determine if there was a possible secretion of steroids during maturation, we added COCs, denuded oocytes and stripped cumulus cells to drops of a steroid-free medium and cultured them for 42 h, after which we analyzed the medium, before and after culture, for the presence of progesterone, estradiol-17beta and testosterone by radioimmunoassay (RIA) analysis. COCs, as well as cumulus cells alone, secreted similar amounts of estradiol (43.3 and 37.5 pg/ml, respectively) and progesterone (4.24 and 4.79 ng/ml, respectively) into the maturation medium. A small amount of estradiol (28.8 pg/ml) was also detected when oocytes were cultured alone. These results indicate that no steroids need to be added to the maturation medium of pig oocytes and that the COCs secrete steroids during maturation. It is possible that the amounts produced by the COCs fulfill any requirement for steroids if these steroids are required for either nuclear or cytoplasmic oocyte maturation. PMID:11991385

  15. Effect of estradiol on oocyte development

    PubMed Central

    Bianco, Katherine; Mahutte, Neal G.; Arici, Aydin; Sakkas, Denny; Taylor, Hugh S.

    2011-01-01

    Objective To determine whether elevated serum estradiol (E2) concentrations in oocyte donors affect assisted reproduction outcome. Method In a retrospective cohort study of 58 consecutive oocyte donation cycles, donors were stratified into 2 groups according to E2 concentration, group 1 (n=32; E2?2000 pg/mL [range 4562000 pg/mL]) and group 2 (n=27; E2>2000 pg/mL [range, 20626957 pg/mL]). Data were analyzed using the t test and ?2 test. Results Donors in group 1 produced significantly less oocytes than donors in group 2 (19.31.7 vs 12.01.4; P<0.001), and recipients of oocytes from group 1 had significantly fewer numbers of embryos available for transfer (10.41.1 vs 6.40.8; P=0.003). However, the mean number (3.3) of embryos transferred and the pregnancy rate were the same in both groups. Conclusion Elevated estradiol concentration in oocyte donors did not affect pregnancy outcome, suggesting that estradiol levels in donors do not affect oocyte development. PMID:19056082

  16. Effects of Resveratrol on Vitrified Porcine Oocytes

    PubMed Central

    Giaretta, Elisa; Spinaci, Marcella; Bucci, Diego; Tamanini, Carlo

    2013-01-01

    Vitrified MII porcine oocytes are characterized by reduced developmental competence, associated with the activation of the apoptotic pathway. Resveratrol (R), a polyphenolic compound present in several vegetal sources, has been reported to exert, among all its other biological effects, an antiapoptotic one. The aim of this study was to determine the effects of R (2?M) on the apoptotic status of porcine oocytes vitrified by Cryotop method, evaluating phosphatidylserine (PS) exteriorization and caspases activation. R was added during IVM (A); 2?h postwarming incubation (B); vitrification/warming and 2?h postwarming incubation (C); all previous phases (D). Data on PS exteriorization showed, in each treated group, a significantly higher (P < 0.05) percentage of live nonapoptotic oocytes as compared with CTR; moreover, the percentage of live apoptotic oocytes was significantly (P < 0.05) lower in all R-treated groups relative to CTR. The results on caspase activation showed a tendency to an increase of viable oocytes with inactive caspases in B, C, and D, while a significant (P < 0.05) increase in A compared to CTR was recorded. These data demonstrate that R supplementation in various phases of IVM and vitrification/warming procedure can modulate the apoptotic process, improving the resistance of porcine oocytes to cryopreservation-induced damage. PMID:24223236

  17. Aven is dynamically regulated during Xenopus oocyte maturation and is required for oocyte survival

    PubMed Central

    O'Shea, L; Fair, T; Hensey, C

    2013-01-01

    We have analyzed the expression and function of the cell death and cell cycle regulator Aven in Xenopus. Analysis of Xenopus Aven expression in oocytes and embryos revealed a band close to the predicted molecular weight of the protein (36?kDa) in addition to two bands of higher molecular weight (46 and 49?kDa), one of which was determined to be due to phosphorylation of the protein. The protein is primarily detected in the cytoplasm of oocytes and is tightly regulated during meiotic and mitotic cell cycles. Progesterone stimulation of oocytes resulted in a rapid loss of Aven expression with the protein levels recovering before germinal vesicle breakdown (GVBD). This loss of Aven is required for the G2M1 cell cycle transition. Aven morpholino knockdown experiments revealed that early depletion of the protein increases progesterone sensitivity and facilitates GVBD, but prolonged depletion of Aven results in caspase-3 activation and oocyte death by apoptosis. Phosphorylated Aven (46?kDa) was found to bind Bcl-xL in oocytes, but this interaction was lost in apoptotic oocytes. Thus, Aven alters progesterone sensitivity in oocytes and is critical for oocyte survival. PMID:24201807

  18. A Miniature Probe for Ultrasonic Penetration of a Single Cell

    PubMed Central

    Wu, Ting; Zhou, Zhaoying; Wang, Qun; Yang, Xing; Xiao, Mingfei

    2009-01-01

    Although ultrasound cavitation must be avoided for safe diagnostic applications, the ability of ultrasound to disrupt cell membranes has taken on increasing significance as a method to facilitate drug and gene delivery. A new ultrasonic resonance driving method is introduced to penetrate rigid wall plant cells or oocytes with springy cell membranes. When a reasonable design is created, ultrasound can gather energy and increase the amplitude factor. Ultrasonic penetration enables exogenous materials to enter cells without damaging them by utilizing instant acceleration. This paper seeks to develop a miniature ultrasonic probe experiment system for cell penetration. A miniature ultrasonic probe is designed and optimized using the Precise Four Terminal Network Method and Finite Element Method (FEM) and an ultrasonic generator to drive the probe is designed. The system was able to successfully puncture a single fish cell. PMID:22412314

  19. The effects of cooling mouse oocytes.

    PubMed

    Sathananthan, A H; Kirby, C; Trounson, A; Philipatos, D; Shaw, J

    1992-04-01

    The effects of cooling and warming on meiotic spindles of mouse oocytes have been assessed by transmission electron microscopy. Intact cumulus-oocyte complexes were immediately cooled from 37 to 15, 4, 0, and -7 degrees C (seeding temperature) for 15 min in a programmed biological freezer and fixed at these temperatures. Other complexes, cooled to these temperatures, were rapidly warmed to 37 degrees C and incubated for 2 hr before fixation at 37 degrees C. Of 334 oocytes assessed at various temperatures, at least 100 were examined for metaphase II spindles. Spindle microtubules completely disappear at 0 and -7 degrees C, while complete or partial depolymerization of microtubules was observed at 4 degrees C. Cooling to 15 degrees C did not cause major disruptions of spindle structure in most oocytes. Chromosomes tended to rotate or clump at lower temperatures but chromosome scatter outside the spindle zone was rarely observed. Centrosomal material was fragmented at 4 degrees C and occasionally at 15 degrees C and was not evident at the spindle poles at 0 and -7 degrees C. Kinetochores were seen at all temperatures. Spindle structure was evidently restored in the majority of oocytes on rewarming at 37 degrees C. Changes in the ooplasm induced by cooling were elongation and disruption of vesicular smooth endoplasmic reticulum, especially between lipid globules and disappearance of fibrillar inclusions. Cortical granule exocytosis was not observed on cooling, while microfilaments were intact. Swelling of membranous organelles was also observed in cumulus cells. Most of the cytoplasmic changes were also reversed on rewarming. The response of mouse oocytes to cooling is compared to that of human oocytes, reported previously. PMID:1627930

  20. Proteomes of Animal Oocytes: What Can We Learn for Human Oocytes in the In Vitro Fertilization Programme?

    PubMed Central

    Virant-Klun, Irma; Krijgsveld, Jeroen

    2014-01-01

    Oocytes are crucial cells for mammalian reproduction, yet the molecular principles underlying oocyte development are only partially understood. Therefore, contemporary proteomic approaches have been used increasingly to provide new insights into oocyte quality and maturation in various species such as mouse, pig, and cow. Especially, animal studies have helped in elucidating the molecular status of oocytes during in vitro maturation and other procedures of assisted reproduction. The aim of this review is to summarize the literature on mammalian oocyte proteome and secretome research in the light of natural and assisted reproduction and on lessons to be learned for human oocytes, which have so far remained inaccessible for proteome analysis. PMID:24804254

  1. Fertilizability of oocytes derived from Holstein cows having different antral follicle counts in ovaries.

    PubMed

    Nagai, Katsuhisa; Yanagawa, Yojiro; Katagiri, Seiji; Nagano, Masashi

    2015-12-01

    In this study, to clarify the relationship between ovarian reserve and oocyte quality, cumulus-oocyte complexes (COCs) were collected repeatedly by ovum pick-up (OPU) from cows with high and low antral follicle counts (AFCs) at short (3-4 days) and long (7 days) intervals, and COC morphologies and oocyte fertilizability were examined. The relationship between AFC and follicular growth after OPU was also investigated. Cows showing AFC of ?30 in at least one OPU session were grouped into the high-AFC group. At a short interval, follicular sizes and COC morphologies were similar between the different AFC groups. However, the normal fertilization rate was higher in the high-AFC group than in the low one, although total penetration rates were similar. At a long interval, the percentage of COCs with poor morphology in the high-AFC group was higher and the normal fertilization rate was lower than in the low one. In the low-AFC group, normal fertilization rates at short and long intervals were similar, and mean follicular size became larger at a long than at a short interval. However, mean follicular sizes at short- and long-interval OPU were similar in the high-AFC group. In conclusion, it is suggested that oocytes derived from cows with high AFC had higher fertilizability than those from cows with low AFC when OPUs were performed at a short (3-4 days) interval. However, oocyte quality in high-AFC cows was impaired by long-interval (7 days) OPU, possibly due to the degradation of follicles. PMID:26588889

  2. 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%.

  3. Genome analyses of single human oocytes.

    PubMed

    Hou, Yu; Fan, Wei; Yan, Liying; Li, Rong; Lian, Ying; Huang, Jin; Li, Jinsen; Xu, Liya; Tang, Fuchou; Xie, X Sunney; Qiao, Jie

    2013-12-19

    Single-cell genome analyses of human oocytes are important for meiosis research and preimplantation genomic screening. However, the nonuniformity of single-cell whole-genome amplification hindered its use. Here, we demonstrate genome analyses of single human oocytes using multiple annealing and looping-based amplification cycle (MALBAC)-based sequencing technology. By sequencing the triads of the first and second polar bodies (PB1 and PB2) and the oocyte pronuclei from same female egg donors, we phase the genomes of these donors with detected SNPs and determine the crossover maps of their oocytes. Our data exhibit an expected crossover interference and indicate a weak chromatid interference. Further, the genome of the oocyte pronucleus, including information regarding aneuploidy and SNPs in disease-associated alleles, can be accurately deduced from the genomes of PB1 and PB2. The MALBAC-based preimplantation genomic screening in in vitro fertilization (IVF) enables accurate and cost-effective selection of normal fertilized eggs for embryo transfer. PMID:24360273

  4. The effect of porcine follicular fluid on the interaction of boar spermatozoa with zona-free hamster ova.

    PubMed Central

    Ramsoondar, J; Downey, B R; Bousquet, D

    1991-01-01

    Boar spermatozoa were cocultured with zona-free hamster ova (eggs) to assess the effects of preovulatory porcine follicular fluid (pFF) in the capacitation medium or gamete coculture (fertilization) medium (pFF; 0, 10 or 40% v/v) on subsequent sperm-egg interaction. Increasing pFF concentrations in the capacitation medium resulted in a progressive decrease in the average numbers of sperm attaching to or penetrating each ovum. When pFF was included in the fertilization medium, but not in the capacitation medium, the average numbers of sperm attaching to or penetrating each ovum and the percentage of ova with sperm attached decreased markedly with increasing pFF concentrations. The percentage of ova with greater than five sperm attached decreased from 84% to 13% and 0% with 0%, 10% and 40% pFF, respectively. Sperm attachment was completely inhibited in approximately 50% of the ova cocultured in 40% pFF. The percentage of ova penetrated by greater than five sperm decreased from 82% to 21% and 7% with 0%, 10% and 40% pFF, respectively. Preincubation of ova in 40% pFF prior to coculture with sperm also resulted in a reduction in sperm attachment and penetration. These results suggest that pFF contains substance(s) that alter the ability of boar spermatozoa to interact with the hamster ovum plasma membrane in vitro. Images Fig. 1. Fig. 2. PMID:1889030

  5. CYTOPLASMIC MICROTUBULAR DYNAMIC AND CHROMATIN ORGANIZATION DURING MAMMALIAN OOCYTE MATURATION

    EPA Science Inventory

    Coordinated alterations in oocyte chromosome and microtubule disposition occur during oogenesis and oocyte maturation in the mammal. imely transitions in meiotic spindle and cytoplasmic microtubules, due to modifications in both the assembly competence of the tubulin pool and nuc...

  6. 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.

  7. Ethical issues in transnational "mail order" oocyte donation.

    PubMed

    Heng, B C

    2006-12-01

    The rising demand for donor oocytes in developed countries has led to what is referred to as transnational or international oocyte donation, or the outsourcing of oocyte donation to poorer countries. In a further twist, frozen sperm from a recipient's partner can also be mailed to a foreign clinic to fertilize donor oocytes, and the resulting embryos are mailed back, cryopreserved, for transfer to the recipient. Among the numerous ethical concerns raised by this practice of mail order oocyte donation, the most obvious are that underprivileged women from poorer countries are often exploited; fertility physicians from richer counties abdicate responsibility for the welfare of donors; and responsibility could become an issue of contention if transmission of disease to the oocyte recipient or congenital defects in offspring born from such oocyte donation were to occur. Moreover, savings from utilizing donors from poorer countries ought to be shared with oocyte recipients. PMID:16999962

  8. Penetrating paediatric thoracic injury.

    PubMed

    Coley, E; Roach, P; Macmillan, A I; West, A T H; Johnston, A M

    2011-09-01

    Paediatric penetrating chest trauma is common in conflict, but rarely seen in peacetime. We describe the successful hospital management of a five year old female civilian casualty with life threatening penetrating thoracic trauma caused by a fragment from an explosive device. PMID:21977715

  9. Follicular penetration and targeting.

    PubMed

    Lademann, Jrgen; 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. PMID:16382687

  10. 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.

  11. Portrait of an oocyte: our obscure origin

    PubMed Central

    Gosden, Roger; Lee, Bora

    2010-01-01

    Oocytes play a pivotal role in the cycle of human life. As we discuss here, after emerging from germline stem cells in the fetus, they grow in a follicular niche in which development is harmonized for timely ovulation and hormone secretion after puberty. Most human oocytes have poor developmental competence and are peculiarly vulnerable to chromosomal malsegregation, especially as women pass the optimal years of fertility and may begin to turn to assisted reproductive technologies (ARTs) and egg donation. Research needs to focus on the molecular factors involved and the environmental niche required for optimal development of oocytes, with the aim of increasing their numbers and quality for ARTs, since these are the factors that so often limit human fertility. PMID:20364095

  12. Oocyte follicle cells association during development of human ovarian follicle. A study by high resolution scanning and transmission electron microscopy.

    PubMed

    Motta, P M; Makabe, S; Naguro, T; Correr, S

    1994-10-01

    Morphodynamics of oocyte follicle cells association during the development of human ovarian follicles were studied by transmission electron microscopy and high resolution scanning electron microscopy including the ODO method. For this study primordial, primary, growing preantral and antral follicles were systematically analysed in a total of 20 adult and fetal (3-8 months and at term) ovaries. In early stages of follicle development (primordial and primary stages) the flattened and/or polyhedral cells, closely associated with the growing oocyte, project an increasing number of microvillous processes. These are in apposition with the oolemma, and form bulbous terminals presenting attachment zones such as zonula adherens, desmosomes and communicating junctions (gap junctions). "Focal contacts" between oolemma, and lateral microvillous extensions of follicle cells were also present. Unusual forms of contact between follicle cell microvilli and oocytes in the early stages of growing primordial and primary follicles were also observed. These consist of long, thin extensions penetrating into the oocyte through deep invaginations of the oolemma. The aid of high resolution SEM of specimens subjected to the ODO method clearly reveals their 3-D arrangement within the ooplasm. They appear as long tortuous microvilli coming very close to the nucleus, and in their course are closely associated with a variety of organelles such as Golgi vesicles, endoplasmic reticulum membranes and nascent forms of smooth endoplasmic reticulum. Using integrated observations by TEM and SEM, there may be as many as 3-5 "intraooplasmic processes" even in only one plane of fracture of an oocyte. Therefore, if the total volume of the oocyte and associated cells is considered, their amounts appear to be higher than previously reported. Thus, they have to be considered as normal devices of deep contact between the ooplasm and associated follicle cell extensions. The presence of such structures within the ooplasm in early developing follicles well coincides with the great increase in volume of the oocyte. Although it is commonly believed that the activation of the growing oocyte may depend on the numerous contacts between the oolemma and follicle cells (mostly via gap junctions), the finding of these additional intraoocytic extensions suggests that they may in someway contribute to the initiation of growth in the human. In fact, these microvilli penetrate deep into the ooplasm, much like a sword in its sheath.(ABSTRACT TRUNCATED AT 400 WORDS) PMID:7880591

  13. In vitro maturation (IVM) of human oocytes.

    PubMed

    Strowitzki, Thomas

    2013-11-01

    In vitro maturation is a technique of assisted reproduction which in contrast to standard IVF or ICSI almost fully avoids hormonal stimulation. Immature oocytes will be fully matured in vitro within 24 h after oocyte collection. The method was introduced in the early nineties and is indicated in patients at high risk for ovarian hyperstimulation. Results are almost comparable to standard techniques. Up to now no elevated risk for fetal malformations has been described. IVM is a suitable alternative in IVF for an exactly defined subgroup of patients, in particular patients with PCOS, but still does not replace standard techniques. PMID:24068296

  14. Fbos, a novel oocyte-specific protein, interacts with proteins important for oocyte development in rainbow trout (Oncorhynchus mykiss)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Oogenesis is characterized by a series of developmentally regulated events, which result in the matured oocyte that can give rise to a new organism after fertilization. Oocyte-specific genes play critical roles in oogenesis; however, the molecular details of oocyte-specific genes are poorly defined....

  15. 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

  16. Intrafollicular influences on human oocyte developmental competence: perifollicular vascularity, oocyte metabolism and mitochondrial function.

    PubMed

    Van Blerkom, J

    2000-07-01

    While genetic and epigenetic factors have been associated with the developmental competence of human oocytes and embryos produced by IVF, how such factors develop or influence the oocyte remain to be explained. This paper reviews evidence which suggests that the degree of perifollicular vascular expansion associated with increased rates of blood flow are developmentally important for the generation of a normal follicle and competent oocyte. The degree of vascular development is follicle specific and differences between follicles might reflect their unique abilities to regulate angiogenic growth factor(s) production by the follicle cells in response to hypoxia. The notion that mitochondrial function in oocytes and early embryos could be influenced by intrafollicular conditions, and that differential patterns of mitochondrial segregation occur in blastomeres during early cleavage, is discussed with respect to the role of these organelles as critical determinants of developmental competence. PMID:11041523

  17. [Abdominal penetrating trauma].

    PubMed

    Kring, Sren; Helligse, Per; Kg, Lise

    2009-06-22

    A 19-year-old female was brought to the Emergency Room as a trauma patient. During a tilting contest she fell off the horse and was penetrated by a spear used for tilting the ring. She was respiratorically as well as haemodynamically stable. The spear was supported but not removed by the paramedics. The spear penetrated the patient near the left iliac crest pointing at the heart. Further investigation at the Emergency Room is described briefly and guidelines for penetrating, impaled foreign bodies in the (thoraco)abdominal region are outlined. PMID:19671404

  18. Sperm and Oocyte Communication Mechanisms Controlling C. elegans Fertility

    PubMed Central

    Han, Sung Min; Cottee, Pauline A.; Miller, Michael A.

    2010-01-01

    During sexual reproduction in many species, sperm and oocyte secrete diffusible signaling molecules to help orchestrate the biological symphony of fertilization. In the Caenorhabditis elegans gonad, bidirectional signaling between sperm and oocyte is important for guiding sperm to the fertilization site and inducing oocyte maturation. The molecular mechanisms that regulate sperm guidance and oocyte maturation are being delineated. Unexpectedly, these mechanisms are providing insight into human diseases, such as amyotrophic lateral sclerosis, spinal muscular atrophy, and cancer. Here we review sperm and oocyte communication in C. elegans and discuss relationships to human disorders. PMID:20034089

  19. 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

  20. Mouse oocyte killing by neutrons: target considerations

    SciTech Connect

    Straume, T.; Dobson, R.L.

    1985-04-01

    Highly radiosensitive primordial mouse oocytes, the principal cells at genetic risk in the female, have been studied using 0.43-MeV neutrons. Analysis of the survival curve (D/sub 37/ = 0.055 Gy) indicates that the diameter of the radiosensitive target (assumed spherical and of unit density) is larger than that of the nucleus but not of the oocyte, implicating a non-nuclear but sub-cellular target. This is consistent with results from /sup 3/H-thymidine incorporated in DNA. Our efforts to identify the extraordinarily radiosensitive lethality target in these primordial oocytes suggest it is the plasma membrane. Monte Carlo calculations for 0.43-MeV neutrons show that at the D/sub 37/ only a single proton recoil will traverse the plasma membrane, consistent with the observed exponential survival curve. A highly sensitive non-DNA target for mouse oocyte killing may importantly influence interpretations of genetic mutation data from mice and their use in estimating genetic risk in humans. 7 refs., 1 fig., 1 tab.

  1. Ca2+ homeostasis regulates Xenopus oocyte maturation.

    PubMed

    Sun, Lu; Hodeify, Rawad; Haun, Shirley; Charlesworth, Amanda; MacNicol, Angus M; Ponnappan, Subramaniam; Ponnappan, Usha; Prigent, Claude; Machaca, Khaled

    2008-04-01

    In contrast to the well-defined role of Ca2+ signals during mitosis, the contribution of Ca2+ signaling to meiosis progression is controversial, despite several decades of investigating the role of Ca2+ and its effectors in vertebrate oocyte maturation. We have previously shown that during Xenopus oocyte maturation, Ca2+ signals are dispensable for entry into meiosis and for germinal vesicle breakdown. However, normal Ca2+ homeostasis is essential for completion of meiosis I and extrusion of the first polar body. In this study, we test the contribution of several downstream effectors in mediating the Ca2+ effects during oocyte maturation. We show that calmodulin and calcium-calmodulin-dependent protein kinase II (CAMK2) are not critical downstream Ca2+ effectors during meiotic maturation. In contrast, accumulation of Aurora kinase A (AURKA) protein is disrupted in cells deprived of Ca2+ signals. Since AURKA is required for bipolar spindle formation, failure to accumulate AURKA may contribute to the defective spindle phenotype following Ca2+ deprivation. These findings argue that Ca2+ homeostasis is important in establishing the oocyte's competence to undergo maturation in preparation for fertilization and embryonic development. PMID:18094360

  2. 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. PMID:7654379

  3. Mouse Oocyte Microinjection, Maturation and Ploidy Assessment

    PubMed Central

    Stein, Paula; Schindler, Karen

    2011-01-01

    Mistakes in chromosome segregation lead to aneuploid cells. In somatic cells, aneuploidy is associated with cancer but in gametes, aneuploidy leads to infertility, miscarriages or developmental disorders like Down syndrome. Haploid gametes form through species-specific developmental programs that are coupled to meiosis. The first meiotic division (MI) is unique to meiosis because sister chromatids remain attached while homologous chromosomes are segregated. For reasons not fully understood, this reductional division is prone to errors and is more commonly the source of aneuploidy than errors in meiosis II (MII) or than errors in male meiosis 1,2. In mammals, oocytes arrest at prophase of MI with a large, intact germinal vesicle (GV; nucleus) and only resume meiosis when they receive ovulatory cues. Once meiosis resumes, oocytes complete MI and undergo an asymmetric cell division, arresting again at metaphase of MII. Eggs will not complete MII until they are fertilized by sperm. Oocytes also can undergo meiotic maturation using established in vitro culture conditions 3. Because generation of transgenic and gene-targeted mouse mutants is costly and can take long periods of time, manipulation of female gametes in vitro is a more economical and time-saving strategy. Here, we describe methods to isolate prophase-arrested oocytes from mice and for microinjection. Any material of choice may be introduced into the oocyte, but because meiotically-competent oocytes are transcriptionally silent 4,5 cRNA, and not DNA, must be injected for ectopic expression studies. To assess ploidy, we describe our conditions for in vitro maturation of oocytes to MII eggs. Historically, chromosome-spreading techniques are used for counting chromosome number 6. This method is technically challenging and is limited to only identifying hyperploidies. Here, we describe a method to determine hypo-and hyperploidies using intact eggs 7-8. This method uses monastrol, a kinesin-5 inhibitor, that collapses the bipolar spindle into a monopolar spindle 9 thus separating chromosomes such that individual kinetochores can readily be detected and counted by using an anti-CREST autoimmune serum. Because this method is performed in intact eggs, chromosomes are not lost due to operator error. PMID:21808228

  4. Sirt1 protects pig oocyte against in vitro aging.

    PubMed

    Ma, Rujun; Zhang, Yu; Zhang, Liang; Han, Jun; Rui, Rong

    2015-09-01

    Sirtuins have been widely reported to be involved in multiple biological processes. However, their function during pig oocyte aging has not been reported yet. Here, we first identify that sirt1 expression is dramatically reduced in pig in vitro-aged oocytes. Furthermore, by confocal scanning and quantitative analysis, we find the increased frequency of spindle defects and chromosome misalignment, disturbed redistribution of cortical granules and mitochondria during oocyte in vitro-aging. Importantly, these aging-associated defective phenotypes can be ameliorated through resveratrol (sirt1 activator) treatment during pig oocyte maturation, providing the evidence for the hypothesis that decreased sirt1 is one of a number of factors contributing to oocyte in vitro-aging. In summary, our data indicate a role for sirt1 in pig oocytes and uncover a striking beneficial effect of sirt1 expression on aged oocytes. PMID:25601632

  5. Selection of Ovine Oocytes by Brilliant Cresyl Blue Staining

    PubMed Central

    Wang, Liqin; Lin, Jiapeng; Huang, Juncheng; Wang, Jing; Zhao, Yuncheng; Chen, Tong

    2012-01-01

    Sheep oocytes derived from the ovaries collected from the slaughterhouse are often used for research on in vitro embryo production, animal cloning, transgenesis, embryonic stem cells, and other embryo biotechnology aspects. Improving the in vitro culture efficiency of oocytes can provide more materials for similar studies. Generally, determination of oocyte quality is mostly based on the layers of cumulus cells and cytoplasm or cytoplasm uniformity and colors. This requires considerable experience to better identify oocyte quality because of the intense subjectivity involved (Gordon (2003), Madison et al. (1992) and De Loos et al. (1992)). BCB staining is a function of glucose-6-phosphate dehydrogenase (G6PD) activity, an enzyme synthesized in developing oocytes, which decreases in activity with maturation. Therefore, unstained oocytes (BCB?) are high in G6PD activity, while the less mature oocytes stains are deep blue (BCB+) due to insuffcient G6PD activity to decolorize the BCB dye. PMID:22675245

  6. Human oocyte chromosome analysis: complicated cases and major pitfalls.

    PubMed

    Rosenbusch, Bernd; Schneider, Michael; Michelmann, Hans Wilhelm

    2008-08-01

    Human oocytes that remained unfertilized in programmes of assisted reproduction have been analysed cytogenetically for more than 20 years to assess the incidence of aneuploidy in female gametes. However, the results obtained so far are not indisputable as a consequence of difficulties in evaluating oocyte chromosome preparations. Because of the lack of guidelines, we decided to summarize for the first time, the possible pitfalls in human oocyte chromosome analysis. Therefore, we screened the material from our previous studies and compiled representative, complicated cases with recommendations for their cytogenetic classification. We point out that maturity and size of the oocyte are important parameters and that fixation artefacts, as well as the particular structure of oocyte chromosomes, may predispose one to misinterpretations. Moreover, phenomena related to oocyte activation and fertilization are illustrated and explained. This compilation may help to avoid major problems in future studies and contribute to a more precise, and uniform assessment of human oocyte chromosomes. PMID:18776643

  7. Selection of ovine oocytes by brilliant cresyl blue staining.

    PubMed

    Wang, Liqin; Lin, Jiapeng; Huang, Juncheng; Wang, Jing; Zhao, Yuncheng; Chen, Tong

    2012-01-01

    Sheep oocytes derived from the ovaries collected from the slaughterhouse are often used for research on in vitro embryo production, animal cloning, transgenesis, embryonic stem cells, and other embryo biotechnology aspects. Improving the in vitro culture efficiency of oocytes can provide more materials for similar studies. Generally, determination of oocyte quality is mostly based on the layers of cumulus cells and cytoplasm or cytoplasm uniformity and colors. This requires considerable experience to better identify oocyte quality because of the intense subjectivity involved (Gordon (2003), Madison et al. (1992) and De Loos et al. (1992)). BCB staining is a function of glucose-6-phosphate dehydrogenase (G6PD) activity, an enzyme synthesized in developing oocytes, which decreases in activity with maturation. Therefore, unstained oocytes (BCB-) are high in G6PD activity, while the less mature oocytes stains are deep blue (BCB+) due to insuffcient G6PD activity to decolorize the BCB dye. PMID:22675245

  8. 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

  9. Checking weld penetration

    NASA Technical Reports Server (NTRS)

    Macfarlane, D. I.

    1979-01-01

    Fused wire in weld root area verifies weld penetration in electron-beam-welded joints. Method could be used in automotive, aircraft, and machinery manufacturing when electron-beam-welds cannot be inspected ultrasonically.

  10. 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. PMID:26453398

  11. 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

  12. Role of focal adhesion kinase in oocyte-follicle communication

    PubMed Central

    McGinnis, Lynda K.; Kinsey, William H.

    2015-01-01

    Germ cells require communication with associated somatic cells for normal gametogenesis as exemplified by the oocyte which interacts with granulosa cells via paracrine factors as well as gap junctions located at sites of contact between these two cell types. The objective of the present study was to define the mechanisms by which cell-cell contact with the oocyte is controlled and determine the extent to which the oocyte actively participates in this association. Focal adhesion kinase (PTK2) was found to be activated at sites of contact between the oocyte and trans-zonal cell processes from the surrounding granulosa cells. In order to determine the functional significance of oocyte-derived PTK2 signaling in oocyte-follicle communication, an oocyte-specific ptk2 knockout was produced through a breeding strategy pairing a floxed ptk2-CAT-eGFP mouse with the Zp3-cre line. Since ptk2-null mice never develop to birth, this represents the first opportunity to define the role of ptk2 in oocytefollicle communication. Ablation of ptk2 within the developing oocyte resulted in lower fertility with reduced numbers of pups, lower rates of blastocyst formation, and reduced cell numbers per blastocyst. Follicles containing ptk2-null oocytes exhibited reduced oocyte diameter, reduced numbers of connexin 37 and 43 foci at the oocyte surface, and impaired dye coupling between oocyte and granulosa cells. These findings are consistent with a model in which PTK2 plays a critical role in establishing or maintaining oocyte-granulosa cell contacts that are essential for gap junction - mediated communication between granulosa cells and the oocyte. PMID:25536210

  13. Expression and localization of cannabinoid receptors in human immature oocytes and unfertilized metaphase-II oocytes.

    PubMed

    Peralta, L; Agirregoitia, E; Mendoza, R; Expsito, A; Casis, L; Matorras, R; Agirregoitia, N

    2011-09-01

    Endocannabinoid anandamide and cannabinoid receptors have been described in some organs of the female reproductive system, but little is known about the expression of these receptors in human oocytes. The aim of the study was to describe the expression of cannabinoid receptors in human oocytes and to investigate their differential distribution at various stages of meiotic resumption in human oocytes. A total of 750 human oocytes from 214 patients were analysed by Western blot, immunocytochemistry and PCR. For this study, oocytes that were not suitable for intracytoplasmic sperm injection (ICSI) (germinal-vesicle and metaphase-I stages), as well as metaphase-II oocytes that had not developed into an embryo after ICSI were used. Western blot analysis revealed the presence of CB1 and CB2 receptor proteins in human oocytes. CB1 and CB2 receptor immunostaining patterns changed during the various stages of meiotic resumption. Localization of CB1 receptor was peripheral at germinal-vesicle stage, homogeneous over the entire oocyte at metaphase I and peripheral at mature metaphase II. CB2 receptor localization was peripheral at germinal-vesicle and metaphase-I stages but homogeneous over the entire cell at metaphase II. This finding suggests a possible role for endocannabinoids, acting via receptors, in the maturation of female gametes and fertilization. The number of couples with sterility problems attending fertility programmes is rising but treatment is not always successful. Important problems associated with failure to conceive remain unresolved because many physiological aspects of human reproduction are still unknown. Endocannabinoids are endogenous chemical compounds that mimic the action of the main psychoactive component of marijuana, delta-9-tetrahydrocannabinol. An endogenous cannabinoid named anandamide has been found in human follicular fluid. Thus, in order to develop knowledge in this field, in the present study we have described the presence of the cannabinoid receptors CB1 and CB2 (the proteins required to mediate the action of the cannabinoids) in the early stages of meiotic resumption of oocytes (the stages before ovulation) and we could postulate that the endocannabinoids could act in the regulation of maturation of oocytes. Our study, together with other studies, indicates that the endocannabinoid system may play a role in human reproduction. PMID:21778114

  14. 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. PMID:25849729

  15. Kinetics of Leptospira interrogans Infection in Hamsters after Intradermal and Subcutaneous Challenge

    PubMed Central

    Coutinho, Mariana L.; Matsunaga, James; Wang, Long-Chieh; de la Pea Moctezuma, Alejandro; Lewis, Michael S.; Babbitt, Jane T.; Aleixo, Jose Antonio G.; Haake, David A.

    2014-01-01

    Background Leptospirosis is a zoonosis caused by highly motile, helically shaped bacteria that penetrate the skin and mucous membranes through lesions or abrasions, and rapidly disseminate throughout the body. Although the intraperitoneal route of infection is widely used to experimentally inoculate hamsters, this challenge route does not represent a natural route of infection. Methodology/Principal Findings Here we describe the kinetics of disease and infection in hamster model of leptospirosis after subcutaneous and intradermal inoculation of Leptospira interrogans serovar Copenhageni, strain Fiocruz L1-130. Histopathologic changes in and around the kidney, including glomerular and tubular damage and interstitial inflammatory changes, began on day 5, and preceded deterioration in renal function as measured by serum creatinine. Weight loss, hemoconcentration, increased absolute neutrophil counts (ANC) in the blood and hepatic dysfunction were first noted on day 6. Vascular endothelial growth factor, a serum marker of sepsis severity, became elevated during the later stages of infection. The burden of infection, as measured by quantitative PCR, was highest in the kidney and peaked on day 5 after intradermal challenge and on day 6 after subcutaneous challenge. Compared to subcutaneous challenge, intradermal challenge resulted in a lower burden of infection in both the kidney and liver on day 6, lower ANC and less weight loss on day 7. Conclusions/Significance The intradermal and subcutaneous challenge routes result in significant differences in the kinetics of dissemination and disease after challenge with L. interrogans serovar Copenhageni strain Fiocruz L1-130 at an experimental dose of 2106 leptospires. These results provide new information regarding infection kinetics in the hamster model of leptospirosis. PMID:25411782

  16. 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

  17. 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.

  18. Autophagic activation in vitrified-warmed mouse oocytes.

    PubMed

    Bang, Soyoung; Shin, Hyejin; Song, Haengseok; Suh, Chang Suk; Lim, Hyunjung Jade

    2014-07-01

    Vitrification involves the use of cryoprotectants (CPAs) and liquid nitrogen (LN2), which may cause osmotic damage and cryoinjury to oocytes. Autophagy is widely recognized as a survival or response mechanism elicited by various environmental and cellular stressors. However, the induction of autophagy in vitrified-warmed oocytes has not been examined. In this work, we investigated whether the vitrification-warming process induces autophagy in mouse oocytes. Metaphase II (MII) oocytes that were vitrified and stored in LN2 for at least 2 weeks were used in the study. In RT-PCR analyses, we observed that several Atg genes such as Atg5, Atg7, Atg12, LC3a (Map1lc3a), LC3b (Map1lc3b), and Beclin1 were expressed in MII mouse oocytes. Slight reduction in mRNA levels of Atg7 and Atg12 in vitrified-warmed oocytes was noted, and expression of these genes was not significantly influenced. Confocal live imaging analysis using oocytes from GFP-LC3 transgenic mice revealed that vitrified-warmed oocytes had a significantly higher number of GFP-LC3 puncta in comparison to fresh oocytes. The expression of BECLIN1 protein was also increased in vitrified-warmed oocytes. Treatment with 3-methyladenine, an inhibitor of autophagy, did not significantly affect the rates of oocyte survival, IVF, and embryonic development after warming and IVF. The results suggest that the observed autophagic activation in vitrified-warmed oocytes is a natural adaptive response to cold stress. Collectively, we show for the first time that vitrified-warmed mouse oocytes exhibit autophagic activation during warming and that this response is not induced by CPA-containing solutions. The induction of autophagy by cold temperature is first reported herein. PMID:24760879

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

    PubMed

    Ortiz, Maria Eugenia; Arias-Torres, Ana Josefina; Zelarayn, 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 F2? (PGF2?) and, to a lesser extent, PGE2, induced meiosis resumption. Oocyte maturation in response to PGF2? was similar to that produced by progesterone (P4). Oocyte response to PGE1 was scarce. Rhinella arenarum oocyte PGF2?-induced maturation showed seasonal variation. From February to June, oocytes presented low sensitivity to PGF2?. 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 PGF2? 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 PGF2? induces germinal vesicle breakdown (GVBD) and meiosis resumption up to metaphase II. In turn, oocytes matured by the action of PGF2? 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 PGF2?. 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

  20. 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.

  1. A potassium current evoked by growth hormone-releasing hormone in follicular oocytes of Xenopus laevis.

    PubMed Central

    Yoshida, S; Plant, S

    1991-01-01

    1. Electrophysiological properties of the growth hormone-releasing hormone (GRH) receptor were studied in Xenopus oocytes with an intact follicle cell layer (i.e. follicular oocytes) by measuring whole-cell current using the two-electrode voltage-clamp method. 2. A slow transient outward current was elicited in oocytes, clamped at -60 mV, by the application of rat GRH but not bovine, porcine, or human GRH. 3. The response to GRH was not suppressed by blockers known to inhibit other endogenous receptors present in follicular Xenopus oocytes; blockers used were timolol (2 microM; beta-adrenergic blocker), theophylline (0.1 mM; purinergic blocker) and atropine (100 nM; muscarinic blocker). 4. The current response evoked by rat GRH occurred in a dose-dependent manner. The concentrations of GRH for threshold and maximum responses were 1 and 100 nM respectively and the estimated EC50 (half-maximal effective concentration) was approximately 7 nM. The amplitude and conductance of the response became larger and the latency, time-to-peak and half-decay time were shortened when the concentration of GRH was increased. 5. The GRH response was reversibly inhibited by a K+ channel blocker, tetraethylammonium+ (TEA+; 20 mM). The reversal potential for the GRH response was around -100 mV and was compatible with the reported value for a K+ current in Xenopus oocytes. Furthermore, a depolarizing shift of 40 mV in the reversal potential was observed when the external K+ concentration was increased from 2 to 10 mM, agreeing with the Nernst equation. In contrast, no significant shift in the reversal potential was observed by changing the external concentration of Na+ or Cl-. 6. The GRH response was not suppressed in oocytes treated with an acetoxy-methyl ester of bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid (BAPTA/AM; 10 microM) which penetrates the cell membrane and chelates internal Ca2+. 7. The GRH response was potentiated by pre-treatment with forskolin (0.4 microM; 5 min), which stimulates adenylate cyclase and increases the internal concentration of adenosine 3',5'-cyclic monophosphate (cyclic AMP). 8. The GRH response was not obtainable when follicle cells surrounding oocytes were removed mechanically with forceps or enzymically with collagenase (i.e. denuded oocytes). The response was also suppressed when gap junctions, which electrically couple follicle cells and the oocyte, were blocked by 1-octanol (1 mM). 9. The first amino acid is considered to be important for the binding of peptide ligands to their receptors.(ABSTRACT TRUNCATED AT 400 WORDS) PMID:1822542

  2. Follicular steroid hormones as markers of oocyte quality and oocyte development potential

    PubMed Central

    Carpintero, Nayara Lpez; Surez, Onica Armijo; Mangas, Carmen Cuadrado; Varea, Carolina Gonzlez; Rioja, Rubn Gmez

    2014-01-01

    CONTEXT: Various components of follicular fluid are suggested as biochemical predictors of oocyte quality. Previous studies of follicular steroid hormone levels have shown disparate results when related with fertilization outcomes. AIM: The objective of the study was to relate the levels of steroid hormones of each individual follicle with oocyte maturation, fertilization results, embryo quality, and pregnancy rates. SETTINGS AND DESIGN: Prospective cohort study in a university hospital. METHODS: In 31 patients, who underwent intracytoplasmic sperm injection, it was performed an ultrasound guided aspiration of follicular fluid of the first two mature follicles from each ovary. Follicular levels of estradiol, progesterone, testosterone, and dehydroepiandrosterone sulfate were measured by chemiluminescent immunoassay. STATISTICAL ANALYSIS: Generalized estimating equation model. RESULTS: In follicular fluids with mature oocyte presence, in normal as well as in failed fertilization, there was a positive correlation between follicular testosterone and progesterone (r = 0.794, P = 0.0001 and r = 0.829, P = 0.0001). Progesterone levels were higher in cases of normal fertilization compared to failed fertilization (P = 0.003). B quality embryos came from oocytes immersed in follicular fluids with higher estradiol values and higher estradiol/progesterone and estradiol/testosterone ratios than those of C quality (P = 0.01; P = 0.0009; P = 0.001). Estradiol levels were higher in patients who achieved pregnancy (P = 0.02). CONCLUSION: The analysis of follicular hormone composition could be considered as an additional tool in oocyte selection. PMID:25395744

  3. [Oocyte removal in conjunction with pelvic microsurgery].

    PubMed

    Ventruba, P; Pilka, L; Cupr, Z; Trvnik, P; Malenovsk, A; Zakov, J

    1991-01-01

    The paper evaluates results of gamete transfer into the oviduct or embryos into the uterus in connection with pelvic microsurgery. Between August 1981-December 1989 225 microsurgical operations have been performed in a stimulated cycle. Ovaries have been stimulated by clomiphene-citrate or in combination with human menopausal gonadotropin. The operation was timed 34 hours after application of human chorionic gonadotropin. In a group of 156 women we recovered at least one oocyte. From 77 gamete transfers into the oviduct we achieved 11 pregnancies, i.e. 14.3 per cent. 46 embryo transfers into the uterus resulted in 5 pregnancies, i.e. 10.9 per cent. From the total number of 16 pregnancies we achieved 5 deliveries, 8 clinic abortions and 3 ectopic pregnancies. The connection of sterility microsurgery with oocyte retrieval increases the success rate of operations without another stress of a patient and has also its economic significance. PMID:1927103

  4. 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

  5. Calcium Signaling enhancement during oocyte maturation

    NASA Astrophysics Data System (ADS)

    Jung, Peter; Ullah, Ghanim; Machaca, Khaled

    2006-03-01

    A Ca2+ signal with a special spatial and temporal characteristic universally removes cell-cycle arrest after fertilization of a mature egg cell. The Ca2+ signal is characterized by a fast rise of intracellular Ca2+ and a slow decay on the time scale of minutes. We use computational modeling of Ca2+ release on the microscale (Ca2+ puffs) and cell-scale in conjunction with experimental knowledge of the changes in the Ca2+ signaling apparatus during oocyte maturation and changing signaling patterns to explore the relationship between organization and sensitivity of IP3 receptors and SERCA pumps and the resulting signaling patterns. We hypothesize that potentiation of the IP3 receptors during oocyte maturation is the main cause for the differentiation in the signaling patterns.

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

    PubMed Central

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

    2015-01-01

    During mammalian oocyte growth, genomic DNA may accumulate DNA double-strand breaks (DSBs) induced by factors such as reactive oxygen species. Recent evidence demonstrated that slight DSBs do not activate DNA damage checkpoint proteins in denuded oocytes. These oocytes, even with DNA DSBs, can resume meiosis and progress to metaphase of meiosis II. Meiotic resumption in oocytes is also controlled by the surrounding cumulus cells; accordingly, we analyzed whether cumulus-cell enclosed oocytes (CEOs) with DNA damage are able to resume meiosis. Compared with DNA-damaged denuded oocytes, we found that meiotic resumption rates of CEOs significantly decreased. To assess the mechanism by which cumulus cells block meiotic resumption in CEOs with DNA DSBs, we treated the cumulus oocyte complex with the gap junction inhibitor carbenoxolone and found that carbenoxolone can rescue the block in CEO meiosis induced by DNA DSBs. Since cumulus cell-synthesized cAMPs can pass through the gap junctions between oocyte and cumulus cell to block oocyte meiosis, we measured the expression levels of adenylate cyclase 1 (Adcy1) in cumulus cells, and G-protein coupled receptor 3 (Gpr3) and phosphodiesterase 3A (Pde3a) in oocytes, and found that the mRNA expression level of Adcy1 increased significantly in DNA-damaged cumulus cells. In conclusion, our results indicate that DNA DSBs promote cAMP synthesis in cumulus cells, and cumulus cAMPs can inhibit meiotic resumption of CEOs through gap junctions. PMID:26575642

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

    PubMed Central

    Choi, Jung Kyu; He, Xiaoming

    2013-01-01

    Background The outbred (as with humans) deer mice have been a useful animal model of research on human behavior and biology including that of the reproductive system. One of the major challenges in using this species is that the yield of oocyte isolation via superovulation is dismal according to the literature to date less than ?5 oocytes per animal can be obtained so far. Objective The goal of this study is to improve the yield of oocyte isolation from outbred deer mice close to that of most laboratory mice by in vitro maturation (IVM) of cumulus-oocyte complexes (COCs). Methods Oocytes were isolated by both superovulation and IVM. For the latter, COCs were obtained by follicular puncture of antral follicles in both the surface and inner cortical layers of ovaries. Immature oocytes in the COCs were then cultured in vitro under optimized conditions to obtain metaphase II (MII) oocytes. Quality of the oocytes from IVM and superovulation was tested by in vitro fertilization (IVF) and embryo development. Results Less than ?5 oocytes per animal could be isolated by superovulation only. However, we successfully obtained 20.32.9 oocytes per animal by IVM (16.02.5) and superovulation (4.31.3) in this study. Moreover, IVF and embryo development studies suggest that IVM oocytes have even better quality than that from superovulation The latter never developed to beyond 2-cell stage as usual while 9% of the former developed to 4-cells. Significance We have successfully established the protocol for isolating oocytes from deer mice with high yield by IVM. Moreover, this is the first ever success to develop in vitro fertilized deer mice oocytes beyond the 2-cell stage in vitro. Therefore, this study is of significance to the use of deer mice for reproductive biology research. PMID:23457518

  8. A rational approach to oocyte cryopreservation.

    PubMed

    Paynter, S J

    2005-05-01

    Reports of clinical pregnancies from cryopreserved human oocytes have been steadily increasing in recent years. However, success in terms of births per thawed oocyte remains poor. A wide variety of freezing techniques has been used lately, but modifications to protocols are made on an empirical basis. Methods of cryopreservation are often poorly described or protocols are not strictly adhered to, resulting in variability of outcome. The first stage of a freezing protocol is exposure to cryoprotectant. If performed inappropriately, such exposure can result in damage due to chemical toxicity and/or osmotic stress. Measurement of cell volume change during exposure to cryoprotectants demonstrates the extent of osmotic stress experienced by that cell. Such measurements have been performed during perfusion of murine and human oocytes with cryoprotectant concentrations commonly used for cryopreservation of these cells. It has been demonstrated that changes in the cryoprotectant type, concentration and temperature of exposure can dramatically affect the extent of cell volume change. Even small changes in duration of exposure to cryoprotectant prior to cooling can result in drastic changes in cellular hydration. Such factors will potentially influence the ability of the cell to survive the stresses experienced during the subsequent stages of the cryopreservation protocol. PMID:15949213

  9. 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 ?M rapamycin/24 h, 47.525.68) or control oocytes (44 h IVM; 42.144.40) significantly increased the development rate and total cell number compared with untreated aged oocytes (68 h IVM, 22.045.68) (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

  10. Immunization of adult hamsters against Clostridium difficile-associated ileocecitis and transfer of protection to infant hamsters.

    PubMed

    Kim, P H; Iaconis, J P; Rolfe, R D

    1987-12-01

    In this investigation, the role of antibodies against Clostridium difficile toxins A and B in protecting hamsters against C. difficile-associated ileocecitis was examined. We also studied the transfer of protection against C. difficile-associated intestinal disease from immunized female hamsters to their infants. Adult female hamsters immunized parenterally with toxoid A or a mixture containing both toxoids A and B were protected against clindamycin-induced C. difficile-associated fatal ileocecitis. On the other hand, hamsters immunized with toxoid B or a broth filtrate from a nontoxigenic strain of C. difficile were not protected against C. difficile-induced ileocecitis. Antibody against the immunizing toxoid could be demonstrated in both the serum and the cecal contents of hamsters. Some infant hamsters from mothers immunized with toxoid A or AB were protected against C. difficile-associated ileocecitis, while infant hamsters from mothers immunized with toxoid B or a nontoxigenic broth filtrate were not protected against disease. Neutralizing antibodies to toxins A and B could be demonstrated in both maternal milk and serum, as well as in infant serum and intestinal contents. Foster-mothering experiments demonstrated that maternal protection of infants against C. difficile-associated ileocecitis was transferred to infant hamsters through breast milk. These results suggest that toxin A may play a more important role in the pathogenesis of C. difficile-associated ileocecitis in hamsters than toxin B. Furthermore, variations in the severity of C. difficile-associated illness in infants and adults may reflect the lack or presence of passively or actively acquired immunity against C. difficile toxins. PMID:3679541

  11. Soil penetrometers and penetrability

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Soil penetrometers are useful tools that measure the penetrability, or strength, of a soil. They can be as simple as a rod or shaft with a blunt or sharp end, or complicated mechanically driven instruments with digital data collection systems. Regardless of their design, soil penetrometers measure s...

  12. Single wall penetration equations

    NASA Technical Reports Server (NTRS)

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

    1991-01-01

    Five single plate penetration equations are compared for accuracy and effectiveness. These five equations are two well-known equations (Fish-Summers and Schmidt-Holsapple), two equations developed by the Apollo project (Rockwell and Johnson Space Center (JSC), and one recently revised from JSC (Cour-Palais). They were derived from test results, with velocities ranging up to 8 km/s. Microsoft Excel software was used to construct a spreadsheet to calculate the diameters and masses of projectiles for various velocities, varying the material properties of both projectile and target for the five single plate penetration equations. The results were plotted on diameter versus velocity graphs for ballistic and spallation limits using Cricket Graph software, for velocities ranging from 2 to 15 km/s defined for the orbital debris. First, these equations were compared to each other, then each equation was compared with various aluminum projectile densities. Finally, these equations were compared with test results performed at JSC for the Marshall Space Flight Center. These equations predict a wide variety of projectile diameters at a given velocity. Thus, it is very difficult to choose the 'right' prediction equation. The thickness of a single plate could have a large variation by choosing a different penetration equation. Even though all five equations are empirically developed with various materials, especially for aluminum alloys, one cannot be confident in the shield design with the predictions obtained by the penetration equations without verifying by tests.

  13. 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.

  14. 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 blood. PMID:23986882

  15. Tumor-penetrating peptides.

    PubMed

    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 ?v 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 blood. PMID:23986882

  16. Protein synthetic patterns in immature and mature human oocytes

    SciTech Connect

    Schultz, G.A.; Gifford, D.J.; Mahadevan, M.M.; Fleetham, J.A.; Taylor, P.J.

    1988-01-01

    A preliminary study of protein synthesis and amino acid transport in human oocytes was initiated. Qualitative patterns or protein synthesis were examined in individual oocytes cultured in medium containing radiolabeled methionine. The protein synthetic profile of immature oocytes, resolved by one-dimensional electrophoresis and fluorography, was observed to change markedly following germinal vesicle breakdown and oocyte maturation. No further differences in the one-dimensional protein synthetic patterns were observed in mature oocytes maintained in culture from 10 hours up to as long as 50 hours. The protein synthetic pattern of follicular cells was observed to be distinct from that of oocytes and was characterized by the predominant synthesis of a polypeptide with Mr = 44,000. Based on the specific activity of the methionine precursor, the absolute rate of synthesis was calculated to be about 50 pg protein/oocyte/hour. Total protein content was measured to be about 150 ng/egg. Competition of methionine uptake by leucine, efflux of radiolabeled methionine from preloaded oocytes into medium containing methionine and uptake of methionine in medium with low sodium ion concentration was observed. These findings are consistent with the presence of an L (leucine-preferring) system for neutral amino acid transport, similar to that in mouse and rabbit eggs. These studies provide basic data for further analysis of oocytes and perhaps preimplantation-stage embryos in the future.

  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. Molecular methods for selection of the ideal oocyte.

    PubMed

    Patrizio, P; Fragouli, E; Bianchi, V; Borini, A; Wells, D

    2007-09-01

    Some recent strategies for identifying the ideal oocyte for insemination in assisted reproduction techniques are reviewed. Established methods of assessing the female gamete, such as morphological evaluation of oocytes and cytogenetic analysis of polar bodies using fluorescence in-situ hybridization, will soon be joined by more advanced cytogenetic methods such as the use of comparative genomic hybridization to improve understanding of oocyte genetics. It seems likely, however, that the greatest advances will originate from the evolution of molecular genetic technologies. The application of microarray technology to individual oocytes and their associated cumulus cells has recently been accomplished, providing a simultaneous assessment of activity for thousands of genes and revealing potential viability markers. Furthermore, improved equipment and optimized methods of mass spectrometry have provided sufficient sensitivity to allow proteomic profiles to be generated from single oocytes and embryos, while metabolomic investigations have searched for indicators of oocyte/embryo quality in spent culture medium. Techniques of this type may ultimately lead to non-invasive tests for oocyte quality revealing previously hidden information concerning both oocyte and embryo developmental competence. Once fully validated, these new approaches are expected to revolutionize oocyte and embryo selection, leading to improved implantation rates and higher probabilities of success using elective single embryo transfer. PMID:17854537

  19. 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

  20. Oocyte morphology predicts outcome of intracytoplasmic sperm injection.

    PubMed

    Serhal, P F; Ranieri, D M; Kinis, A; Marchant, S; Davies, M; Khadum, I M

    1997-06-01

    To examine the influence of cytoplasmic morphology on the success rate of intracytoplasmic sperm injection (ICSI), the morphology of 837 metaphase II oocytes was assessed after cumulus stripping. The main abnormalities detected were excessive granularity, cytoplasmic inclusions such as vacuoles, smooth endoplasmic reticulum clustering and refractile bodies. Microinjection was performed in 538 oocytes with normal cytoplasm, 142 out of 161 with excessive granularity and 112 out of 138 with cytoplasmic inclusions. Very poor oocytes were not injected. No difference was found in fertilization rate. The embryos achieved cleaved normally and a similar number of good quality embryos among the three groups was noted. The outcome of transfer of embryos derived solely from normal oocytes (group A: 72 patients, 183 embryos) was compared with those from oocytes with cytoplasmic abnormalities (group B: 34 patients, 85 embryos). In group A, 17 clinical pregnancies (24% per patient, implantation rate 10%) were established. In group B, only one clinical pregnancy (3% per patient, implantation rate 1%) was established, from the transfer of embryos derived from oocytes with homogeneous granularity of the cytoplasm. No pregnancy resulted following the transfer of embryos from eggs with cytoplasmic inclusions. The difference was statistically significant. The outcome of ICSI is dependent on the quality of the oocytes retrieved. Normal fertilization and early embryo development were achieved in oocytes with abnormal cytoplasm morphology, but the resulting embryos failed to demonstrate the same implantation potential as those derived from oocytes with normal cytoplasm. PMID:9222015

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

    PubMed

    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

  2. The efficacy of melatonin administration on oocyte quality.

    PubMed

    Bat?o?lu, A Serta; Sahin, U?ra?; Grlek, Beril; Oztrk, Nazire; Unsal, Evrim

    2012-02-01

    The aim of the study was to evaluate the efficacy of melatonin administration on oocyte quality in women underwent in vitro fertilization (IVF) cycles. Eighty-five women undergoing IVF cycles were randomized in two groups during IVF-embryo transfer (ET) procedure, 40 women with melatonin treatment (A) and 45 women without melatonin treatment (B). Primary endpoint was the number of morphologically mature oocytes retrieved (MII oocytes). Secondary endpoints were fertilization rate per number of mature oocytes, embryo quality and pregnancy rate. There were no differences between two groups according to age, and peak estradiol levels. The mean number of oocytes (15.33 vs. 14.27) and the mean number of mature oocytes did not differ between the two groups (12.63 vs. 10.94), whereas the percentage of mature oocytes (M2/oocytes retrieved) was significantly different in melatonin-treated group (p < 0.05). Fertilization rate (72.75 vs. 71.16) did not differ between the two groups. The mean number of class 1 embryos resulted higher in the group A (3.28 vs. 2.53) (p < 0.05). Clinical pregnancy rate was in tendency higher in the group treated with melatonin, although the differences did not reach statistical significance. Melatonin is likely to improve oocyte and embryo quality in women undergoing IVF or intracytoplasmic sperm insemination (ICSI). PMID:21770829

  3. 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

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

    PubMed

    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

  5. 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.

  6. Melatonin-enhanced hyperactivation of hamster sperm.

    PubMed

    Fujinoki, Masakatsu

    2008-11-01

    The effects of melatonin on reproductive function were examined using hamster spermatozoa. When 1 pM to 1 microM melatonin was added to the mTALP medium, hyperactivation was significantly enhanced. Antagonists and agonists of the melatonin receptor (i.e., MT1 and MT2) were added to the medium. Luzindole, an MT1 and MT2 competitive antagonist, significantly inhibited melatonin-induced hyperactivation, whereas the MT2-specific antagonists, 4-phenyl-2-propionamidotetralin and N-pentanoyl-2-benzyltryptamine, had no effect. Moreover, hyperactivation was significantly enhanced when non-specific agonists, such as 6-chloromelatonin and 2-iodomelatonin, were added to the medium. 8-Methoxy-2-propionamidotetralin, which is a strong MT2 agonist and a weak MT1 agonist, significantly increased hyperactivation, although the effect was weak. Therefore, it is likely that melatonin enhances sperm hyperactivation via the MT1 receptor. PMID:18715981

  7. Role of acidification elicited by sialylation and sulfation of zona glycoproteins during oocyte maturation in porcine sperm-zona pellucida interactions.

    PubMed

    Lay, Khin Mar; Oshiro, Ryuko; Arasaki, Chiemi; Ashizawa, Koji; Tatemoto, Hideki

    2011-12-01

    The porcine zona pellucida (ZP) undergoes biochemical changes during the final phase of maturation prior to fertilization. The present study was conducted to elucidate whether the acidification of ZP glycoproteins during porcine oocyte maturation influences sperm-ZP interactions. Two-dimensional gel electrophoresis clearly demonstrated that ZP acidification occurred in accordance with the sialylation and sulfation of ZP glycoproteins in oocytes matured for 44 h. The increases in the incidences of sperm penetration and polyspermy with the progress of the IVM culture period were significantly suppressed by ZP desialylation on treatment with neuraminidase as a consequence of reductions in the number of sperm bound to ZPs and the acrosome reaction (AR) in ZP-bound sperm (P<0.05). In contrast, the blocking of ZP sulfation by NaClO(3) treatment during IVM markedly reduced the incidence of polyspermy with no inhibitory effect on penetration, but the number of sperm bound to ZPs and the rate of AR-inducing sperm were decreased to the same level as in desialylated oocytes. The results indicate that ZP sulfation influences sperm-ZP interactions in a ZP sialylation-independent manner. Moreover, sialylation and sulfation were not associated with a protective proteolytic modification of the ZP matrix before fertilization. These findings suggest that ZP acidification elicited by the sialylation and sulfation of ZP glycoproteins during oocyte maturation contributes to the porcine ZP acquiring the capacity to accept sperm. PMID:21897057

  8. In Vitro Oocyte Maturation and Preantral Follicle Culture from the Luteal-Phase Baboon Ovary Produce Mature Oocytes1

    PubMed Central

    Xu, Min; Fazleabas, Asgerally T.; Shikanov, Ariella; Jackson, Erin; Barrett, Susan L.; Hirshfeld-Cytron, Jenny; Kiesewetter, Sarah E.; Shea, Lonnie D.; Woodruff, Teresa K.

    2010-01-01

    Female cancer patients who seek fertility preservation but cannot undergo ovarian stimulation and embryo preservation may consider 1) retrieval of immature oocytes followed by in vitro maturation (IVM) or 2) ovarian tissue cryopreservation followed by transplantation or in vitro follicle culture. Conventional IVM is carried out during the follicular phase of menstrual cycle. There is limited evidence demonstrating that immature oocyte retrieved during the luteal phase can mature in vitro and be fertilized to produce viable embryos. While in vitro follicle culture is successful in rodents, its application in nonhuman primates has made limited progress. The objective of this study was to investigate the competence of immature luteal-phase oocytes from baboon and to determine the effect of follicle-stimulating hormone (FSH) on baboon preantral follicle culture and oocyte maturation in vitro. Oocytes from small antral follicle cumulus-oocyte complexes (COCs) with multiple cumulus layers (42%) were more likely to resume meiosis and progress to metaphase II (MII) than oocytes with a single layer of cumulus cells or less (23% vs. 3%, respectively). Twenty-four percent of mature oocytes were successfully fertilized by intracytoplasmic sperm injection, and 25% of these developed to morula-stage embryos. Preantral follicles were encapsulated in fibrin-alginate-matrigel matrices and cultured to small antral stage in an FSH-independent manner. FSH negatively impacted follicle health by disrupting the integrity of oocyte and cumulus cells contact. Follicles grown in the absence of FSH produced MII oocytes with normal spindle structure. In conclusion, baboon luteal-phase COCs and oocytes from cultured preantral follicles can be matured in vitro. Oocyte meiotic competence correlated positively with the number of cumulus cell layers. This study clarifies the parameters of the follicle culture system in nonhuman primates and provides foundational data for future clinical development as a fertility preservation option for women with cancer. PMID:21123815

  9. Inhibition of bovine sperm-oocyte fusion by a monoclonal antibody recognising the TEC-2 epitope on bovine oocytes.

    PubMed

    Gougoulidis, T; Trounson, A; Dowsing, A

    1999-10-01

    The TEC-2 antigenic determinant is a carbohydrate epitope located on a glycoprotein carrier molecule. In the mouse, this epitope is expressed on the zona pellucida and plasma membrane of the oocyte and is associated with the ZP2 glycoprotein and involved in the secondary sperm receptor mechanism. On the bovine oocyte expression is confined to the plasma membrane. The aim of this study was to determine the role the TEC-2 epitope plays during fertilization in the bovine species using the monoclonal antibody TEC-02. Incubating oocytes with the TEC-02 antibody prior to fertilization inhibited cleavage in a dose-dependent manner-the cleavage rate decreased as the concentration of the antibody increased. Significantly more sperm were bound to oocytes exposed to TEC-02 (12 sperm/oocyte) compared to oocytes that were not incubated with the antibody (4 sperm/oocyte). Oocytes treated with the TEC-02 antibody had a 7.5 +/- 3.2% fusion rate and no cortical granule exocytosis compared with oocytes not exposed to the antibody, with 86.5 +/- 5.8% of sperm-oocyte fusions and release of cortical granules. The block to sperm-oocyte fertilization observed in the pretreated group was overcome using intracytoplasmic sperm injection as the method of fertilization that bypassed the fusion process. Although sperm were binding to the oolemma these results suggest that fusion was not occurring and this may be due to the antibody occupying TEC-2 epitope sites involved in the fusion process. In conclusion, the TEC-2 epitope seems to be involved in sperm-oocyte interaction in the bovine species and appears to be involved specifically during the fusion events of fertilization. PMID:10471477

  10. Photoperiodic regulation of the hamster testis: dependence on circadian rhythms.

    PubMed Central

    Eskes, G A; Zucker, I

    1978-01-01

    The testes of hamsters exposed to short days (10 hr of light per day) regress within 13 weeks. Administration of 7.5% deuterium oxide to hamsters lengthens the period of free running circadian activity rhythms by 2.2% and prevents testicular regression during short-day exposure. This consistent with predictions derived from an external coincidence model for photoperiodic time measurement: Deuterium oxide changes phase relationships between the light-dark cycle and the circadian system, the hamster's daily photosensitive phase is stimulated with light during short days, and the testes remain large. Conservation of the period of circadian rhythms within narrow limits has adaptive significance for hamster photoperiodism and for the occurrence and phasing of the annual reproductive cycle. Images PMID:273214

  11. NEONATAL CHIORDECONE EXPOSURE ALTERS BEHAVIORAL SEX DIFFERENTIATION IN FEMALE HAMSTERS

    EPA Science Inventory

    The present study was designed in order to determine if exposure to the weakly estrogenic pesticide Chlordecone during a critical period of behavioral sex differentiation of the brain could masculinize and defeminize the behavior of female hamsters.

  12. Direct Peptide Interaction with Surface Glycosaminoglycans Contributes to the Cell Penetration of Maurocalcine*

    PubMed Central

    Ram, Narendra; Aroui, Sonia; Jaumain, Emilie; Bichraoui, Hicham; Mabrouk, Kamel; Ronjat, Michel; Lortat-Jacob, Hugues; De Waard, Michel

    2008-01-01

    Maurocalcine (MCa), initially identified from a tunisian scorpion venom, defines a new member of the family of cell penetrating peptides by its ability to efficiently cross the plasma membrane. The initiating mechanistic step required for the cell translocation of a cell penetrating peptide implicates its binding onto cell surface components such as membrane lipids and/or heparan sulfate proteoglycans. Here we characterized the interaction of wild-type MCa and MCa K20A, a mutant analogue with reduced cell-penetration efficiency, with heparin (HP) and heparan sulfates (HS) through surface plasma resonance. HP and HS bind both to MCa, indicating that heparan sulfate proteoglycans may represent an important entry route of the peptide. This is confirmed by the fact that (i) both compounds bind with reduced affinity to MCa K20A and (ii) the cell penetration of wild-type or mutant MCa coupled to fluorescent streptavidin is reduced by about 50% in mutant Chinese hamster ovary cell lines lacking either all glycosaminoglycans (GAGs) or just HS. Incubating MCa with soluble HS, HP, or chondroitin sulfates also inhibits the cell penetration of MCa-streptavidin complexes. Analyses of the cell distributions of MCa/streptavidin in several Chinese hamster ovary cell lines show that the distribution of the complex coincides with the endosomal marker Lyso-Tracker red and is not affected by the absence of GAGs. The distribution of MCa/streptavidin is not coincident with that of transferrin receptors nor affected by a dominant-negative dynamin 2 K44A mutant, an inhibitor of clathrin-mediated endocytosis. However, entry of the complex is greatly diminished by amiloride, indicating the importance of macropinocytosis in MCa/streptavidin entry. It is concluded that (i) interaction of MCa with GAGs quantitatively improves the cell penetration of MCa, and (ii) GAG-dependent and -independent MCa penetration rely similarly on the macropinocytosis pathway. PMID:18603532

  13. 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.

  14. Hydrogen sulfide donor protects porcine oocytes against aging and improves the developmental potential of aged porcine oocytes.

    PubMed

    Krejcova, Tereza; Smelcova, Miroslava; Petr, Jaroslav; Bodart, Jean-Francois; Sedmikova, Marketa; Nevoral, Jan; Dvorakova, Marketa; Vyskocilova, Alena; Weingartova, Ivona; Kucerova-Chrpova, Veronika; Chmelikova, Eva; Tumova, Lenka; Jilek, Frantisek

    2015-01-01

    Porcine oocytes that have matured in in vitro conditions undergo the process of aging during prolonged cultivation, which is manifested by spontaneous parthenogenetic activation, lysis or fragmentation of aged oocytes. This study focused on the role of hydrogen sulfide (H2S) in the process of porcine oocyte aging. H2S is a gaseous signaling molecule and is produced endogenously by the enzymes cystathionine-?-synthase (CBS), cystathionine-?-lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (MPST). We demonstrated that H2S-producing enzymes are active in porcine oocytes and that a statistically significant decline in endogenous H2S production occurs during the first day of aging. Inhibition of these enzymes accelerates signs of aging in oocytes and significantly increases the ratio of fragmented oocytes. The presence of exogenous H2S from a donor (Na2S.9H2O) significantly suppressed the manifestations of aging, reversed the effects of inhibitors and resulted in the complete suppression of oocyte fragmentation. Cultivation of aging oocytes in the presence of H2S donor positively affected their subsequent embryonic development following parthenogenetic activation. Although no unambiguous effects of exogenous H2S on MPF and MAPK activities were detected and the intracellular mechanism underlying H2S activity remains unclear, our study clearly demonstrates the role of H2S in the regulation of porcine oocyte aging. PMID:25615598

  15. Hydrogen Sulfide Donor Protects Porcine Oocytes against Aging and Improves the Developmental Potential of Aged Porcine Oocytes

    PubMed Central

    Krejcova, Tereza; Smelcova, Miroslava; Petr, Jaroslav; Bodart, Jean-Francois; Sedmikova, Marketa; Nevoral, Jan; Dvorakova, Marketa; Vyskocilova, Alena; Weingartova, Ivona; Kucerova-Chrpova, Veronika; Chmelikova, Eva; Tumova, Lenka; Jilek, Frantisek

    2015-01-01

    Porcine oocytes that have matured in in vitro conditions undergo the process of aging during prolonged cultivation, which is manifested by spontaneous parthenogenetic activation, lysis or fragmentation of aged oocytes. This study focused on the role of hydrogen sulfide (H2S) in the process of porcine oocyte aging. H2S is a gaseous signaling molecule and is produced endogenously by the enzymes cystathionine-?-synthase (CBS), cystathionine-?-lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (MPST). We demonstrated that H2S-producing enzymes are active in porcine oocytes and that a statistically significant decline in endogenous H2S production occurs during the first day of aging. Inhibition of these enzymes accelerates signs of aging in oocytes and significantly increases the ratio of fragmented oocytes. The presence of exogenous H2S from a donor (Na2S.9H2O) significantly suppressed the manifestations of aging, reversed the effects of inhibitors and resulted in the complete suppression of oocyte fragmentation. Cultivation of aging oocytes in the presence of H2S donor positively affected their subsequent embryonic development following parthenogenetic activation. Although no unambiguous effects of exogenous H2S on MPF and MAPK activities were detected and the intracellular mechanism underlying H2S activity remains unclear, our study clearly demonstrates the role of H2S in the regulation of porcine oocyte aging. PMID:25615598

  16. Bisphenol-A and human oocyte maturation in vitro

    PubMed Central

    Machtinger, Ronit; Combelles, Catherine M.H.; Missmer, Stacey A.; Correia, Katharine F.; Williams, Paige; Hauser, Russ; Racowsky, Catherine

    2013-01-01

    STUDY QUESTION Does exposure to bisphenol-A (BPA) affect the maturation of human oocytes in vitro? SUMMARY ANSWER There was a doseresponse association of BPA exposure with altered human oocyte maturation in vitro. WHAT IS KNOWN ALREADY There is widespread exposure of the general population to BPA. BPA has been detected in the human follicular fluid. Animal studies have shown that BPA exposure is associated with maturation arrest and spindle abnormalities in maturing oocytes. STUDY DESIGN, SIZE, DURATION A randomized trial, using 352 clinically discarded oocytes from 121 patients. PARTICIPANTS/MATERIALS, SETTING, METHODS The study population was drawn from patients undergoing IVF/ICSI cycles in our program at Brigham and Women's Hospital from March 2011 to April 2012. Oocytes from only one cycle for each patient were included in the study. Cycles with at least two germinal vesicle stage oocytes were included with random allocation of one oocyte to culture for 30 h without BPA and remaining sibling oocytes to medium-containing BPA (20, 200 ng/ml or 20 g/ml). Oocytes were fixed and labeled for tubulin, actin and chromatin and examined with immunofluorescence and confocal microscopy. Oocytes were assessed for meiotic stage (n = 292), and those at metaphase II (MII, n = 175) were further classified according to their spindle configurations and patterns of chromosome alignment. McNemar's test was used to compare dichotomized maturation status. Generalized estimating equations were used to account for the correlation between oocytes from the same woman and for the spindle analysis. MAIN RESULTS AND THE ROLE OF CHANCE As the BPA dose increased, there was a decrease in the percentage of oocytes that progressed to MII (P = 0.002) and increases in the percentage of oocytes that were degenerated (P = 0.01) or that had undergone spontaneous activation (P = 0.007). Among MII oocytes, as the BPA dose increased, there was a significant trend (by test for trend) for a decreased incidence of bipolar spindles (P < 0.0001) and aligned chromosomes (P = 0.02). LIMITATIONS, REASONS FOR CAUTION Although we used sibling oocytes to overcome potential confounders, such as infertility diagnosis and maternal age, additional studies with a larger number of oocytes are required to confirm the present results. Having access only to clinically discarded oocytes, we were limited to evaluating only those oocytes that failed to mature in vivo despite having been exposed to gonadotrophin stimulation and the ovulatory trigger of HCG. WIDER IMPLICATIONS OF THE FINDINGS To our knowledge, this is the first study investigating the effect of BPA on oocyte meiotic maturation, spindle morphology and chromosome alignment in human oocytes. Together with prior animal studies, the data support the negative influences of BPA on cell cycle progression, spindle architecture and chromosome organization during oocyte maturation. Furthermore, the increased rates of abnormal maturation in oocytes exposed to BPA may be relevant to our understanding of the decrease in fertility reported in the last decades. STUDY FUNDING/COMPETING INTEREST(S) This study was funded by the NIEHS Center Grant Pilot Project (P30-ES000002). R.M. was sponsored by a fellowship from the Environmental Health Fund, Israel and by the Frederick L. Hisaw Endowment, Harvard School of Public Health. There are no conflicts of interest. TRIAL REGISTRATION NUMBER n/a. PMID:23904465

  17. Lactose ingestion in the adult golden hamster (Mesocricetus auratus).

    PubMed

    DiBattista, D

    1991-03-01

    Adult mammals generally demonstrate a lower preference for the disaccharide sugar lactose than for any other common sugar, and because adults typically have low levels of the intestinal enzyme lactase, lactose ingestion may cause gastrointestinal distress. The lactose intake of adult golden hamsters was examined in three experiments; the main findings were: (a) hamsters allowed to choose between tap water and lactose solutions (maximum concentration = 32% weight/volume) over a 20-day period showed a clear preference for lactose solutions and ingested substantial quantities of lactose (up to 3 g/100 g body weight/day) without noticeable adverse effects; (b) hamsters consuming a single diet with lactose added (maximum concentration = 50%) over a period of days ingested up to 3.42 g/100 g body weight/day of lactose without noticeable adverse effects; (c) both hamsters with prior exposure to lactose solutions and those without such exposure consumed similar amounts of 32% lactose solution over an 8-day period, suggesting that hamsters' lactose intake does not depend on the occurrence of adaptation. It is suggested that the fermentative capacity of the hamster's pregastric pouch may underlie this animal's unusual tolerance for lactose. PMID:2032461

  18. 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.

  19. 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.

  20. Hormonal control of cholesterol cholelithiasis in the female hamster.

    PubMed

    Ayyad, N; Cohen, B I; Mosbach, E H; Mikami, T; Mikami, Y; Ohshima, A

    1995-07-01

    Male golden Syrian hamsters from Sasco form cholesterol gallstones when fed a lithogenic diet; in contrast, female hamsters are resistant to stones when fed the identical diet. Upon addition of the synthetic androgen, methyl-testosterone, to the diet, the incidence of cholesterol gallstones in female hamsters increased from 0% to 40% after 3 weeks and from 0% to 86% after 6 weeks. Cholesterol cholelithiasis remained high in the males. Biliary cholesterol and phospholipid levels were elevated in the females fed the hormone and approached those of the males. The cholesterol saturation of bile in the females increased from 36% to 75% after 3 weeks and from 54% to 109% after 6 weeks. In addition, an appreciable proportion of the cholesterol in the bile of female hamsters was now present in the form of vesicles. The bile acid composition was significantly altered by methyltestosterone even though the total bile acid concentration did not change; the bile acid composition of the female hamsters approached that of the males. The glycine to taurine ratio of the bile acids was drastically reduced by methyltestosterone in the females and to a lesser extent in males. In summary, in female hamsters the addition of methyltestosterone to the lithogenic diet induced cholesterol gallstones, elevated total biliary phospholipid and cholesterol, altered the bile acid composition, and changed the distribution of cholesterol from micelles to vesicles. The data obtained so far do not enable us to define the precise mechanism of action of methyltestosterone. PMID:7595072

  1. Behaviour of cytoplasmic organelles and cytoskeleton during oocyte maturation.

    PubMed

    Mao, Luna; Lou, Hangying; Lou, Yiyun; Wang, Ning; Jin, Fan

    2014-03-01

    Assisted reproduction technology (ART) has become an attractive option for infertility treatment and holds tremendous promise. However, at present, there is still room for improvement in its success rates. Oocyte maturation is a process by which the oocyte becomes competent for fertilization and subsequent embryo development. To better understand the mechanism underlying oocyte maturation and for the future improvement of assisted reproduction technology, this review focuses on the complex processes of cytoplasmic organelles and the dynamic alterations of the cytoskeleton that occur during oocyte maturation. Ovarian stimulation and in-vitro maturation are the major techniques used in assisted reproduction technology and their influence on the organelles of oocytes is also discussed. Since the first birth by assisted reproduction treatment was achieved in 1978, numerous techniques involved in assisted reproduction have been developed and have become attractive options for infertility treatment. However, the unsatisfactory success rate remains as a main challenge. Oocyte maturation is a process by which the oocyte becomes competent for fertilization and subsequent embryo development. Oocyte maturation includes both nuclear and cytoplasmic maturation. Nuclear maturation primarily involves chromosomal segregation, which has been well studied, whereas cytoplasmic maturation involves a series of complicated processes, and there are still many parts of this process that remain controversial. Ovarian stimulation and in-vitro maturation (IVM) are the major techniques of assisted reproduction. The effect of ovarian stimulation or IVM on the behaviour of cell organelles of the oocyte has been postulated as the reason for the reduced developmental potential of in-vitro-produced embryos. To further understanding of the mechanism of oocyte maturation and future improvement of assisted reproduction treatment, the complex events of cytoplasmic organelles and the cytoskeleton that occur during oocyte maturation and the influence of ovarian stimulation and IVM on these organelles are described in this review. PMID:24444815

  2. Oocyte morphology from primordial to early tertiary follicles of yak.

    PubMed

    Yu, S J; Yong, Y H; Cui, Y

    2010-10-01

    The objective of this study was to investigate the developmental morphology of yak oocytes from the primordial follicle to the tertiary follicle. Yak oocytes from resting primordial (n = 6), activated primordial (n = 12), primary (n = 9), secondary (n = 7) and early tertiary (n = 5) follicles were processed and analysed by light and transmission electron microscopy. The resting primordial follicular oocyte was characterized by relatively smooth surface on the oolemma, the accumulation of free and organelle-related smooth (SER) and rough endoplasmic reticulum (RER), round or oval mitochondria, and polyribosomes on the surface of the RER and throughout the ooplasm. The activated primordial follicular oocyte was dominated by numerous coated pits and coated vesicles on the oolemma, and round mitochondria. Up to the secondary follicular stage the oocyte displayed an increase in the number of microvilli, polyribosome, Golgi complexes and mitochondria with distinct cristae. During the secondary follicular stage, formation of the zona pellucida, development of a desmosome-like connection between the oocyte and the granulosa cells, formation of the cortical granules in the oocyte and elongated mitochondria in nearly all oocytes were seen. In the early tertiary follicular oocyte, the perivitelline space was present and a decrease in free SER and RER in the ooplasm occurred; finally, the nucleus migrated from an eccentric to a peripheral location. In conclusion, the growth of the yak oocyte is associated with the relocation and modulation of a number of cytoplasmic organelles as well as the development of oocyte-specific structures such as the zona pellucida, desmosome-like connection and cortical granules. PMID:20059745

  3. Detection of genes associated with developmental competence of bovine oocytes.

    PubMed

    Nemcova, Lucie; Jansova, Denisa; Vodickova-Kepkova, Katerina; Vodicka, Petr; Jeseta, Michal; Machatkova, Marie; Kanka, Jiri

    2016-03-01

    The developmental competence of oocytes is acquired progressively during folliculogenesis and is linked to follicular size. It has been documented that oocytes originating from larger follicles exhibit a greater ability to develop to the blastocyst stage. The differences in cytoplasmic factors such as mRNA transcripts could explain the differences in oocyte developmental potential. We used bovine oligonucleotide microarrays to characterize differences between the gene expression profiles of germinal vesicle stage (GV) oocytes with greater developmental competence from medium follicles (MF) and those with less developmental competence from small follicles (SF). After normalizing the microarray data, our analysis found differences in the level of 60 transcripts (?1.4 fold), corresponding to 49 upregulated and 11 downregulated transcripts in MF oocytes compared to SF oocytes. The gene expression data were classified according to gene ontology, the majority of the genes were associated with the regulation of transcription, translation, the cell cycle, and mitochondrial activity. A subset of 16 selected genes was validated for GV oocytes by quantitative real-time RT-PCR; significant differences (P?0.01) were found in the level of TAF1A, MTRF1L, ATP5C1, UBL5 and MAP3K13 between the MF and SF oocytes. After maturation the transcript level remained stable for ATP5F1, BRD7, and UBL5 in both oocyte categories. The transcript level of another 13 genes substantially dropped in the MF and/or SF oocytes. It can be concluded that the developmental competence of bovine oocytes and embryos may be a quantitative trait dependent on small changes in the transcription profiles of many genes. PMID:26811294

  4. 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.

  5. Penetrating eye injuries.

    PubMed

    Patel, B C

    1989-03-01

    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

  6. 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.

  7. 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

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

    PubMed Central

    Gonca, Sheyla; Gn, Ismet; Ovayolu, Ali; ?ilfeler, Dilek; Sofuo?lu, Kenan; zdamar, zkan; Yilmaz, Ali; Tunali, Glden

    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 Students 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 hand, daily gonadotropin dose (290.9 79.9 and 273.4 74.4, P = 0.034) and total gonadotropin doses (2545 1031.8 and 2247.7 901.9, P = 0.004) were significantly higher in Group 1. The pregnancy rate was significantly higher in Group 1 (29.1% and 19.4%, P = 0.041). No correlation was observed between the number of oocyte and pregnancy (r = 0.082, P = 0.107). Conclusions: The number of aspirated follicles during IVF treatment being higher than the collected number of oocyte leads to a statistically significant fall in the pregnancy rates. There is no correlation between the number of oocyte and pregnancy. PMID:25126190

  9. Regulation of oocyte maturation in fish.

    PubMed

    Nagahama, Yoshitaka; Yamashita, Masakane

    2008-06-01

    A period of oocyte growth is followed by a process called oocyte maturation (the resumption of meiosis) which occurs prior to ovulation and is a prerequisite for successful fertilization. Our studies using fish models have revealed that oocyte maturation is a three-step induction process involving gonadotropin (LH), maturation-inducing hormone (MIH), and maturation-promoting factor (MPF). LH acts on the ovarian follicle layer to produce MIH (17alpha, 20beta-dihydroxy-4-pregnen-3-one, 17alpha, 20beta-DP, in most fishes). The interaction of ovarian thecal and granulosa cell layers (two-cell type model), is required for the synthesis of 17alpha,20beta-DP. The dramatic increase in the capacity of postvitellogenic follicles to produce 17alpha,20beta-DP in response to LH is correlated with decreases in P450c17 (P450c17-I) and P450 aromatase (oP450arom) mRNA and increases in the novel form of P450c17 (P450c17-II) and 20beta-hydroxysteroid dehydrogenase (20beta-HSD) mRNA. Transcription factors such as Ad4BP/SF-1, Foxl2, and CREB may be involved in the regulation of expression of these steroidogenic enzymes. A distinct family of G-protein-coupled membrane-bound MIH receptors has been shown to mediate non-genomic actions of 17alpha, 20beta-DP. The MIH signal induces the de novo synthesis of cyclin B from the stored mRNA, which activates a preexisting 35 kDa cdc2 kinase via phosphorylation of its threonine 161 by cyclin-dependent kinase activating kinase, thus producing the 34 kDa active cdc2 (active MPF). Upon egg activation, MPF is inactivated by degradation of cyclin B. This process is initiated by the 26S proteasome through the first cut in its NH(2) terminus at lysine 57. PMID:18482399

  10. 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.

  11. Periscope For Viewing Weld Penetration

    NASA Technical Reports Server (NTRS)

    Gordon, Stephen S.; Marman, Jonathan M.

    1988-01-01

    Periscope enables viewing of weld joint from inside cylindrical duct to determine when weld penetration occurs. Supplies steady stream of inert gas to shield joint. Device used to calibrate and evaluate techniques for sensing weld penetration.

  12. 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.

  13. 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

  14. Investigations into implantation failure in oocyte-donation recipients.

    PubMed

    Barri, Pedro N; Coroleu, Buenaventura; Clua, Elisabet; Tur, Rosa; Boada, Montserrat; Rodriguez, Ignacio

    2014-01-01

    In recent decades, the Western world has been experiencing a societal trend to prioritize the professional careers of women who postpone motherhood to about 40 years of age, when, unfortunately, natural reproductive potential declines. This is the reason why these women increasingly find it necessary to resort to oocyte donation to have a child. Thanks to the young age of the donors, the efficacy of oocyte donation is the highest of all assisted reproduction treatments and pregnancy rates achieved with this technique exceed 50%. Moreover, the large registries from ESHRE and ASRM show live birth rates close to this figure. However, there are patients who experience repeated failures in several oocyte-donation cycles, and so far oocyte-donation repeated implantation failure has not been clearly defined. This study analysed the results obtained from 2531 oocyte-donation cycles carried out in 1990 patients and defines oocyte-donation repeated implantation failure as failure to implant with more than two embryo transfers and more than four high-grade embryos transferred. This study observed this condition in 140 oocyte recipients (7%). Also, oocyte cohort size, uterine factors and systemic thrombophilias as important aetiological factors were identified were to offer new therapeutic strategies to patients. PMID:24268727

  15. In Vitro Growth and Maturation of Vitrified-Warmed Bovine Oocytes Collected from Early Antral Follicles

    PubMed Central

    HIRAO, Yuji; SOMFAI, Tams; NARUSE, Kenji

    2013-01-01

    Abstract. Cryopreservation of growing oocytes enriches the choice of timing and location of artificial embryo production. However, completion of oocyte growth after warming is crucial when using such cryopreserved oocytes. Our research objective was to develop a sequential system that incorporates cryopreservation of growing bovine oocytes and their subsequent in vitro growth. Oocyte-granulosa cell complexes with a mean oocyte diameter of approximately 100 m were vitrified-warmed and then cultured for 14 days. The percentage of surviving oocytes following cryopreservation and 14-day culture was approximately 80%. More than half of the surviving oocytes were capable of maturing to metaphase II after in vitro maturation; the rate was comparable to that of control oocytes grown in vitro without cryopreservation. Taken together, the combined protocols for vitrification-warming of growing oocytes and subsequent in vitro growth can produce oocytes capable of undergoing meiotic maturation. PMID:24126072

  16. RNA synthesis in pig follicular oocytes. Autoradiographic and cytochemical study.

    PubMed

    Motlk, J; Kopecn, V; Trvnik, P; Pivko, J

    1984-01-01

    RNA synthesis in pig oocytes was studied using autoradiography and silver staining of the nucleolus organizing region. Both methods confirmed that oocytes from the smallest follicles (0.5-0.7 mm in diam.) very intensely synthesize nuclear and nucleolar RNA. The nucleolar area of oocytes originating from follicles of 1.6-2.2 mm in diam. was labelled mainly on its periphery. After short pulse labelling (15 min) of oocytes from follicles of 5-6 mm in diam. only the nucleoplasm was labelled. The nucleolus had no significant labelling. The possibility that labelling of the compact nucleolus after a longer pulse represents migration of the newly synthesized nuclear RNA into the compact nucleolus, is discussed. The quantity of silver-positive material in dictyate oocytes significantly decreased as pig follicles enlarged in diam. from 2 mm to 5-6 mm. PMID:6235877

  17. [Oocyte maturation. From animal experiments to human reproduction techniques].

    PubMed

    Lefvre, B

    1996-01-01

    The control of the oocyte growth and maturation are complex processus implicating endocrines (gonadotropines) and paracrine factors (growth factors, cytokines, steroids...), the paracrine regulation being under the endocrine control. The control of the cytoplasmic and nuclear maturation is realized by substances going through gap junctions from the follicular cells to the oocyte. Two of these substances, which are present in the follicular fluid, are responsible for maintaining the meiotic arrest: cyclic AMP and purines. Hormones or growth factors, which are able to affect the oocyte maturation both in vivo and in vitro, when they interact with their receptors stimulate enzymes which in turn stimulate the release of second messengers. Actually, the whole process that is oocyte maturation followed by fertilization in vitro does not give good results. This difficulty does not permit yet the freezing of immature oocytes which could be a solution in some cases encountered in Medically Assisted Procreation. PMID:8924956

  18. Spindles, mitochondria and redox potential in ageing oocytes.

    PubMed

    Eichenlaub-Ritter, U; Vogt, E; Yin, H; Gosden, R

    2004-01-01

    Studies of human oocytes obtained from women of advanced reproductive age revealed that spindles are frequently aberrant, with chromosomes sometimes failing to align properly at the equator during meiosis I and II. Chromosomal analyses of donated and spare human oocytes and cytogenetic and molecular studies on the origin of trisomies collectively suggest that errors in chromosome segregation during oogenesis increase with advancing maternal age and as the menopause approaches. Disturbances in the fidelity of chromosome segregation, especially at anaphase I, leading to aneuploidy are prime causes of reduced developmental competence of embryos in assisted reproduction, as well as being responsible for the genesis of genetic disease. This review provides an overview of spindle formation and chromosome behaviour in mammalian oocytes. Evidence of a link between abnormal mitochondrial function in oocytes and somatic follicular cells, and finally disturbances in chromosome cohesion and segregation, and cell cycle control in aged mammalian oocytes, are also discussed. PMID:14759287

  19. Peroxynitrite deteriorates oocyte quality through disassembly of microtubule organizing centers.

    PubMed

    Khan, Sana N; Shaeib, Faten; Thakur, Mili; Jeelani, Roohi; Awonuga, Awoniyi O; Goud, Pravin T; Abu-Soud, Husam M

    2016-02-01

    Previous theoretical studies have suggested that utilization of 3-D imaging to acquire morphologic parameters of meiotic spindles may be useful in infertility related procedures as an assessment of oocyte quality. However, our results show that treatment of oocytes with increasing concentrations of peroxynitrite (ONOO(-)) caused a dramatic alteration in spindle shape in which morphologic parameters are not measurable or are uninformative in terms of oocyte quality. Metaphase II mouse oocytes (n=520) were treated with increasing concentrations of ONOO(-), after which all oocytes were fixed and subjected to indirect immunofluorescence. Oocyte quality was assessed by alterations in the microtubule-organizing center (MTOC), pericentrin location, microtubule morphology, and chromosomal alignment. In untreated oocytes, pericentrin is primarily assembled utilizing the acentrosomal MTOC, which appears as a condensation at both spindle poles. The spindle has a symmetrical pointed barrel shape, assembled around the chromosomal plate at the spindle equator. Oocytes treated with low concentrations of ONOO(-) (<2.5?M) showed shortening of the spindle apparatus, while pericentrin scatters from a tight condensation to a dispersed cluster around each spindle pole. At higher ONOO(-) concentrations (>2.5?M) the central attachments between microtubules are strained and bend or unevenly break, and the MTOC proteins are further dispersed or undetectable. Peroxynitrite mediated MTOC damage, which deranges the chromosomal scaffold at the time of assembly and separation, caused the deterioration in oocyte quality. These results provide a link between reactive oxygen species and poor reproductive outcomes and elucidate the underlying etiology, which could be used as a superior biomarker for oocyte quality compared to existing assessment tools. PMID:26746586

  20. Oocyte ultrastructure in bovine primordial to early tertiary follicles.

    PubMed

    Fair, T; Hulshof, S C; Hyttel, P; Greve, T; Boland, M

    1997-04-01

    The aim of the present study was to describe in detail the changes occurring in the cytoplasmic ultrastructure of the bovine oocyte from the onset of growth in the primordial follicle until the completion of growth in the tertiary follicle. Bovine oocytes from primordial, primary, secondary and early to mid-antral follicles were processed and analysed by light and transmission electron microscopy. The primordial follicular oocyte was characterized by numerous coated pits on the oolemma and the accumulation of free and organelle-related smooth (SER) and rough (RER) endoplasmic reticulum, round mitochondria and Golgi complexes around the nucleus, which was located slightly off centre. Up to the secondary follicular stage the oocyte displayed an increase in the number of microvilli, elongated mitochondria and Golgi complexes. During the secondary follicular stage, formation of the zona pellucida, development of gap junctions between the oocyte and the granulosa cells, formation of the cortical granules in the oocyte and reduction in the number of coated pits on the oolemma were seen. In the tertiary follicular oocyte up to 100 microm in diameter, the number of Golgi complexes and lipid droplets increased and the organelles were dislocated to the deep cortical region. During the final growth of the oocyte up to >120 microm, the organelles were dislocated further to the peripheral region, the extent of the free SER and RER compartments were reduced, the number of individual cortical granules increased, hooded mitochondria became abundant and the perivitelline space developed. In conclusion, the growth of the bovine oocyte is associated with the relocation and modulation of a number of cytoplasmic organelles as well as the development of oocyte specific structures such as the zona pellucida and cortical granules. PMID:9108198

  1. Selective carboxyl methylation of structurally altered calmodulins in Xenopus oocytes.

    PubMed

    Desrosiers, R R; Romanik, E A; O'Connor, C M

    1990-12-01

    The eucaryotic protein carboxyl methyltransferase specifically modifies atypical D-aspartyl and L-isoaspartyl residues which are generated spontaneously as proteins age. The selectivity of the enzyme for altered proteins in intact cells was explored by co-injecting Xenopus laevis oocytes with S-adenosyl-L-[methyl-3H]methionine and structurally altered calmodulins generated during a 14-day preincubation in vitro. Control experiments indicated that the oocyte protein carboxyl methyltransferase was not saturated with endogenous substrates, since protein carboxyl methylation rates could be stimulated up to 8-fold by increasing concentrations of injected calmodulin. The oocyte protein carboxyl methyltransferase showed strong selectivities for bovine brain and bacterially synthesized calmodulins which had been preincubated in the presence of 1 mM EDTA relative to calmodulins which had been preincubated with 1 mM CaCl2. Radioactive methyl groups were incorporated into base-stable linkages with recombinant calmodulin as well as into carboxyl methyl esters following its microinjection into oocytes. This base-stable radioactivity most likely represents the trimethylation of lysine 115, a highly conserved post-translational modification which is present in bovine and Xenopus but not in bacterially synthesized calmodulin. Endogenous oocyte calmodulin incorporates radioactivity into both carboxyl methyl esters and into base-stable linkages following microinjection of oocytes with S-adenosyl-[methyl-3H]methionine alone. The rate of oocyte calmodulin carboxyl methylation in injected oocytes is calculated to be similar to that of lysine 115 trimethylation, suggesting that the rate of calmodulin carboxyl methylation is similar to that of calmodulin synthesis. At steady state, oocyte calmodulin contains approximately 0.0002 esters/mol of protein, which turn over rapidly. The results suggest the quantitative significance of carboxyl methylation in the metabolism of oocyte calmodulin. PMID:2123492

  2. 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. PMID:23382023

  3. 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

  4. 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

  5. Penetrating Fire Extinguisher

    NASA Technical Reports Server (NTRS)

    1985-01-01

    When Feecon Corporation, a manufacturer of fire protection systems, needed a piercing nozzle for larger aircraft, they were assisted by Kennedy Space Center who provided the company with a fire extinguisher with a hard pointed tip that had been developed in case of an orbiter crash landing. The nozzle can penetrate metal skins of aircraft, trains, etc. Feecon obtained a license and now markets its cobra ram piercing nozzle to airport firefighters. Its primary advantage is that the nozzle can be held in one spot during repeated blows of the ram. *This product has been discontinued and is no longer commercially available.

  6. Proteomic Analysis of Chinese Hamster Ovary Cells

    PubMed Central

    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

    2013-01-01

    In order to complement the recent genomic sequencing of Chinese hamster ovary (CHO) cells, proteomic analysis was performed on CHO including the cellular proteome, secretome, and glycoproteome using tandem mass spectrometry (MS/MS) of multiple fractions obtained from gel electrophoresis, multi-dimensional 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 a 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 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. 504 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

  7. Humoral and cellular response to infection with Echinostoma revolutum in the golden hamster, Mesocricetus auratus.

    PubMed

    Mabus, J; Huffman, J E; Fried, B

    1988-06-01

    Laboratory hamsters (Mesocricetus auratus) were infected with Echinostoma revolutum (Trematoda). Immunoelectrophoretic studies of hamster serum showed no demonstrable antibody response to E. revolutum. Histopathologic examination of intestinal tissue of infected hamsters showed erosion of intestinal villi and lymphocytic infiltration as the primary host response. Spleens from infected hamsters were hyperplastic during the first 3 weeks of infection and atrophic from 4 to 8 weeks postinfection. Hamsters were unable to acquire a resistance to E. revolutum infection. Lack of resistance was demonstrated in hamsters where the parasite infection was no longer detected based on the absence of eggs in the faeces; these hamsters were then reinfected. Hamsters treated with the anthelmintic oxyclozanide were also reinfected with E. revolutum. PMID:3397514

  8. 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.

  9. Daily hoarding opportunity entrains the pacemaker for hamster activity rhythms.

    PubMed

    Rusak, B; Mistlberger, R E; Losier, B; Jones, C H

    1988-12-01

    The effects on activity rhythms of a daily 30 min opportunity to leave the home cage and hoard seeds from an open field were assessed in Syrian hamsters housed in continuous dim illumination. Six of ten hamsters responded with clear entrainment of their activity rhythms to the hoarding opportunity, as demonstrated by responses to phase shifts and by the onset phase of subsequent freerunning rhythms. No entrainable component separate from the freerunning rhythm was ever observed. Two hamsters showed phase shifts in response to the hoarding opportunity, but they did not meet the criteria for stable entrainment, and two did not respond with noticeable changes in rhythmicity. Ablations of the suprachiasmatic nuclei (SCN) were attempted in three hamsters that had entrained stably to the hoarding time. The effects of partial lesions in two animals indicated that the entrained rhythm was controlled by the light-entrainable pacemaker represented by the SCN. The one animal with an apparently complete lesion, however, developed a clear, but irregular, increase in activity in anticipation of the daily hoarding time. SCN ablation apparently unmasked an oscillator system separate from the SCN and susceptible to entrainment by a nonphotic cue. The oscillator mechanism affected by daily hoarding opportunities in hamsters appears to be tightly coupled to the SCN pacemaker, in contrast to the system in rats that is synchronized by daily feeding schedules. PMID:3244126

  10. Functional microangiopathy in alloxan-treated Syrian hamsters.

    PubMed

    Colantuoni, A; Cimini, V; Coppini, G; Bertuglia, S

    1988-03-01

    Intraperitoneally injected alloxan determined long term hyperglycemia in a group of Syrian hamsters (35 hyperglycemic hamsters); transitory hyperglycemia, with recovery of normal blood glucose concentration but impairment of glucose tolerance test, was observed in a second group of alloxan-treated animals (70 normoglycemic hamsters). Microvascular permeability by fluorescent microscopy technique, capillary basement membrane thickening and pancreatic islet B, A, and D cell degranulation by computer-assisted microdensitometry were studied in Syrian hamsters at different intervals (30, 40, 60, 90, and 120 days) after intraperitoneal alloxan administration. Hyperglycemic groups showed increased permeability of venous microvasculature to high molecular weight dextran in 50%, 71.4%, and 100% of animals studied at 30, 40, and 60, 90, 120 days from treatment, respectively; indeed, they revealed pancreatic islet B cell degranulation and no capillary basement membrane thickening. Normoglycemic groups presented increased venular leakage in 28.5%, 42.8%, 71.4%, and 100% of animals investigated at 40, 60, 90, and 120 days after treatment, respectively; moreover, they showed moderate pancreatic islet B cell degranulation and no capillary basement membrane thickening. In conclusion, more severe microvascular alterations seemed to be related to more severe impairment of glucose metabolism and to longer duration of diabetes; even in normoglycemic hamsters with pathological glucose tolerance test, enhanced permeability developed. PMID:3286553

  11. 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.

  12. 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.

  13. 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.

  14. Penetration in GTA welding

    SciTech Connect

    Heiple, C.R.; Burgardt, P.

    1990-01-01

    The size and shape of the weld bead produced in GTA welding depends on the magnitude and distribution of the energy incident on the workpiece surfaces as well as the dissipation of that energy in the workpiece. The input energy is largely controllable through the welding parameters selected, however the dissipation of that energy in the workpiece is less subject to control. Changes in energy dissipation can produce large changes in weld shape or penetration. Heat transport away from the weld pool is almost entirely by conduction, but heat transport in the weld pool is more complicated. Heat conduction through the liquid is an important component, but heat transport by convection (mass transport) is often the dominant mechanism. Convective heat transport is directional and changes the weld pool shape from that produced by conduction alone. Surface tension gradients are often the dominant forces driving fluid flow in GTA weld pools. These gradients are sensitive functions of weld pool chemistry and the energy input distribution to the weld. Experimental and theoretical work conducted primarily in the past decade has greatly enhanced our understanding of weld pool fluid flow, the forces which drive it, and its effects on weld pool shape. This work is reviewed here. While less common, changes in energy dissipation through the unmelted portion of the workpiece can also affect fusion zone shape or penetration. These effects are also described. 41 refs., 9 figs.

  15. Monolithic ballasted penetrator

    DOEpatents

    Hickerson, Jr., James P. (Cedar Crest, NM); Zanner, Frank J. (Sandia Park, NM); Baldwin, Michael D. (Albuquerque, NM); Maguire, Michael C. (Worcester, MA)

    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.

  16. 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.

  17. Polarity and asymmetry during mouse oogenesis and oocyte maturation.

    PubMed

    Kloc, Malgorzata; Ghobrial, Rafik M; Borsuk, Ewa; Kubiak, Jacek Z

    2012-01-01

    Cell polarity and asymmetry play a fundamental role in embryo development. The unequal segregation of determinants, cues, and activities is the major event in the differentiation of cell fate and function in all multicellular organisms. In oocytes, polarity and asymmetry in the distribution of different molecules are prerequisites for the progression and proper outcome of embryonic development. The mouse oocyte, like the oocytes of other mammals, seems to apply a less stringent strategy of polarization than other vertebrates. The mouse embryo undergoes a regulative type of development, which permits the full rectification of development even if the embryo loses up to half of its cells or its size is experimentally doubled during the early stages of embryogenesis. Such pliability is strongly related to the proper oocyte polarization before fertilization. Thus, the molecular mechanisms leading to the development and maintenance of oocyte polarity must be included in any fundamental understanding of the principles of embryo development. In this chapter, we provide an overview of current knowledge regarding the development and maintenance of polarity and asymmetry in the distribution of organelles and molecules in the mouse oocyte. Curiously, the mouse oocyte becomes polarized at least twice during ontogenesis; the question of how this phenomenon is achieved and what role it might play is addressed in this chapter. PMID:22918799

  18. 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

  19. 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

  20. Homeobox gene expression in human oocytes and cleaving embryos

    SciTech Connect

    Verlinsky, O.; Morozov, G.; Gindilis, V.

    1994-09-01

    Homeobox gene expression in human preimplantation development has not been established. The expression of POU-domain homeobox gene Oct-3 was reported only in mouse oocytes and cleaving embryos. We used reverse transcriptase-polymerase chain reaction (RT-PCR) with the design of primer sets to span an intron to investigate the homeobox gene mRNA presence in human oocytes and pre-embryos. RT-PCR from normal and unfertilized oocytes, cleaving normal and triploid embryos were cloned, sequenced, and analyzed for the presence of homeobox sequences. The presence of mRNA of homeoboxes HoxA4 and HoxA7 was demonstrated: HoxA4 was present in normal and unfertilized oocytes, and also in 4-cell embryos; HoxA7 was present in normal oocytes and cleaving triploid embryos. Semiquantitative experiments using a second round of PCR and an intron spanning primer set specifying to HoxA4 revealed minute quantities of HoxA4 mRNA also in 6-cell embryos but not in 9-cell embryos and early morula. These data are consistent with the hypothesis that HoxA4 mRNA is present in the oocytes and persists in early embryogenesis. Future research will be needed to investigate the spectrum of homeobox gene expression in human oocytes and pre-embryos.

  1. Selective carboxyl methylation of structurally altered calmodulins in Xenopus oocytes

    SciTech Connect

    Desrosiers, R.R.; Romanik, E.A.; O'Connor, C.M. )

    1990-12-05

    The eucaryotic protein carboxyl methyltransferase specifically modifies atypical D-aspartyl and L-isoaspartyl residues which are generated spontaneously as proteins age. The selectivity of the enzyme for altered proteins in intact cells was explored by co-injecting Xenopus laevis oocytes with S-adenosyl-L-(methyl-3H)methionine and structurally altered calmodulins generated during a 14-day preincubation in vitro. Control experiments indicated that the oocyte protein carboxyl methyltransferase was not saturated with endogenous substrates, since protein carboxyl methylation rates could be stimulated up to 8-fold by increasing concentrations of injected calmodulin. The oocyte protein carboxyl methyltransferase showed strong selectivities for bovine brain and bacterially synthesized calmodulins which had been preincubated in the presence of 1 mM EDTA relative to calmodulins which had been preincubated with 1 mM CaCl2. Radioactive methyl groups were incorporated into base-stable linkages with recombinant calmodulin as well as into carboxyl methyl esters following its microinjection into oocytes. This base-stable radioactivity most likely represents the trimethylation of lysine 115, a highly conserved post-translational modification which is present in bovine and Xenopus but not in bacterially synthesized calmodulin. Endogenous oocyte calmodulin incorporates radioactivity into both carboxyl methyl esters and into base-stable linkages following microinjection of oocytes with S-adenosyl-(methyl-3H)methionine alone. The rate of oocyte calmodulin carboxyl methylation in injected oocytes is calculated to be similar to that of lysine 115 trimethylation, suggesting that the rate of calmodulin carboxyl methylation is similar to that of calmodulin synthesis. At steady state, oocyte calmodulin contains approximately 0.0002 esters/mol of protein, which turn over rapidly.

  2. 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

  3. Mutagenic effects of ionizing radiation on immature rat oocytes.

    PubMed

    Asakawa, Jun-ichi; Kamiguchi, Yujiroh; Kamiya, Kenji; Nakamura, Nori

    2014-10-01

    Estimates of genetic risks from radiation delivered to humans are derived largely from mouse studies. In males, the target is spermatogonia and a large amount of information is available. In contrast, in females, immature oocytes are the target, but extrapolations from mice to humans are not very definitive because immature mouse oocytes are highly sensitive to radiation and die by apoptosis, which is not the case in humans. Since mouse offspring derived from surviving immature oocytes have to date not shown any signs of mutation induction, two alternative hypotheses are proposed: 1. Apoptotic death effectively eliminates damaged oocytes in mice and therefore human immature oocytes may be highly mutable; and 2. Immature oocytes are inherently resistant to mutation induction and apoptotic death is not relevant to mutagenesis. To test these hypotheses, rat immature oocytes, which are not as sensitive as those in mice to radiation-induced apoptosis were exposed to 2.5 Gy of gamma rays and the offspring were examined using a two-dimensional DNA analysis method. Screening of a total of 2.26 million DNA fragments, we identified 32 and 18 mutations in the control and exposed groups, respectively. Of these, in the two groups, 29 and 14 mutations were microsatellite mutations, two and one were base changes, and one and three were deletions. Among the four deletions most relevant to radiation exposure, only one was possibly derived from the irradiated dam (but not determined) and three were paternal in origin. Although the number of mutations was small, the results appear to support the second hypothesis and indicate that immature oocytes are generally less sensitive than mature oocytes to mutation induction. PMID:25229977

  4. Androgens promote the acquisition of maturation competence in bovine oocytes

    PubMed Central

    MAKITA, Miho; MIYANO, Takashi

    2015-01-01

    Recent studies in mice suggest that androgens are important for normal follicle development. However, there have been few reports concerning the action of androgens in the growth of oocytes from large animals. The purpose of this study was to determine the roles of androgens in bovine oocyte growth in vitro. Oocyte-granulosa cell complexes (OGCs) collected from 0.4−0.7 mm early antral follicles were cultured for 14 days with 17β-estradiol (E2) and a non-aromatizable androgen, dihydrotestosterone (DHT). We also examined the ability of an androgen receptor (AR) inhibitor, hydroxyflutamide, to antagonize the effect of androgens on the oocytes. During growth culture, the OGC structures collapsed in the medium with DHT alone, while in the presence of E2, the OGC structures were maintained. In the medium with both androgens and E2, the mean diameter of oocytes was increased from 95 μm to around 120 μm, larger than those grown with E2 alone (115 μm). Also in the maturation culture, oocytes grown with androgens (A4 or DHT) and E2 showed higher percentages of metaphase II oocytes (63% or 69%, respectively) than those grown with E2 alone (32%). Moreover, these maturation rates were decreased by hydroxyflutamide in a dose-dependent manner. Immunostaining showed that ARs were expressed in oocytes and granulosa cells in early antral follicles, and the nuclei of granulosa cells showed intense AR expression. In conclusion, although E2 supports the OGC structure, additional androgens promote oocyte growth and their acquisition of meiotic competence via AR during in vitro growth culture. PMID:25754240

  5. 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 ?-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 ?-tubulin polypeptide and one ?-tubulin polypeptide (?/?-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 ?-tubulin. The mutations affect chaperone-dependent folding and assembly of the ?/?-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

  6. A Syrian Golden Hamster Model Recapitulating Ebola Hemorrhagic Fever

    PubMed Central

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

    2013-01-01

    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. PMID:23045629

  7. Effect of (+)-gossypol on fertility in male hamsters.

    PubMed

    Waller, D P; Bunyapraphatsara, N; Martin, A; Vournazos, C J; Ahmed, M S; Soejarto, D D; Cordell, G A; Fong, H H; Russell, L D; Malone, J P

    1983-01-01

    (+)-Gossypol was isolated from the bark of Thespesia populnea and tested for its ability to inhibit the fertility of male hamsters. Male hamsters of proven fertility were treated orally for 54 days with 40 mg/kg of (+)-gossypol, 40 mg/kg of racemic gossypol, or 5% gum acacia (vehicle control) and were mated with estrous female hamsters during the fourth and seventh weeks of treatment. Both the control and the (+)-gossypol-treated animals exhibited normal fertility throughout the experiment. The racemic gossypol-treated animals were infertile when evaluated during both the fourth and seventh weeks of treatment. Morphologic examination of the testicular tissue could not explain the loss of fertility. These data demonstrate the inability of (+)gossypol to decrease male fertility and suggest that the activity of racemic gossypol may be due primarily to the presence of the (-) optical isomer. PMID:6618998

  8. Small GTPases and formins in mammalian oocyte maturation: cytoskeletal organizers

    PubMed Central

    Kwon, Sojung

    2011-01-01

    The maturation process of mammalian oocytes accompanies an extensive rearrangement of the cytoskeleton and associated proteins. As this process requires a delicate interplay between the cytoskeleton and its regulators, it is often targeted by various external and internal adversaries that affect the congression and/or segregation of chromosomes. Asymmetric cell division in oocytes also requires specific regulators of the cytoskeleton, including formin-2 and small GTPases. Recent literature providing clues regarding how actin filaments and microtubules interact during spindle migration in mouse oocytes are highlighted in this review. PMID:22384410

  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. PMID:24750650

  10. Analysis of nuclear reprogramming following nuclear transfer to Xenopus oocyte.

    PubMed

    Jullien, Jerome

    2015-01-01

    Germinal vesicle of stage V-VI Xenopus Laevis oocytes (at the prophase I stage of meiosis) can be used to transplant mammalian nuclei. In this type of interspecies nuclear transfer no cell division occurs and no new cell types are generated. However, the transplanted nuclei undergo extensive transcriptional reprogramming. Here, it is first explained how to carry out transplantation of multiple mammalian cell nuclei to Xenopus oocytes. It is then described how to perform RT-qPCR, Western Blot, Chromatin Immunoprecipitation, and live imaging analysis to monitor transcriptional reprogramming of the nuclei transplanted to oocytes. PMID:25287339

  11. 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

  12. Hamster thecal cells express muscle characteristics

    SciTech Connect

    Self, D.A.; Schroeder, P.C.; Gown, A.M.

    1988-08-01

    Contraction of the follicular wall about the time of ovulation appears to be a coordinated event; however, the cells that mediate it remain poorly studied. We examined the theca externa cells in the wall of hamster follicles for the presence of a functional actomyosin system, both in developing follicles and in culture. We used a monoclonal antibody (HHF35) that recognizes the alpha and gamma isoelectric variants of actin normally found in muscle, but not the beta variant associated with non-muscle sources, to evaluate large preovulatory follicles for actin content and composition. Antibody staining of sectioned ovaries showed intense circumferential reactivity in the outermost wall of developing follicles. Immunoblots from two-dimensional gels of theca externa lysates demonstrated the presence of the two muscle-specific isozymes of actin. Immunofluorescence of cultured follicular cells pulse-labeled with (3H) thymidine (for autoradiographic detection of DNA replication) revealed the presence, in many dividing cells, of actin filaments aligned primarily along the longitudinal axis of the cells. In cultures exposed to the calcium ionophore A23187 (10(-4) M) for varying periods (5 min to 1 h), contraction of many individual muscle-actin-positive cells was observed. Immunofluorescence of these cells, fixed immediately after ionophore-induced contraction, revealed compaction of the actin filaments. Our findings demonstrate that the cells of the theca externa contain muscle actins from an early stage and that these cells are capable of contraction even while proliferating in subconfluent cultures. They suggest that follicular growth may include a naturally occurring developmental sequence in which a contractile cell type proliferates in the differentiated state.

  13. Water penetration study

    NASA Technical Reports Server (NTRS)

    Lockwood, H. E.

    1973-01-01

    Nine film-filter combinations have been tested for effectiveness in recording water subsurface detail when exposed from an aerial platform over a typical water body. An experimental 2-layer positive color film, a 2-layer (minus blue layer) film, a normal 3-layer color film, a panchromatic black-and-white film, and an infrared film with selected filters were tested. Results have been tabulated to show the relative capability of each film-filter combination for: (1) image contrast in shallow water (0 to 5 feet); (2) image contrast at medium depth (5 to 10 feet); (3) image contrast in deep water (10 feet plus); (4) water penetration; maximum depth where detail was discriminated; (5) image color (the spectral range of the image); (6) vegetation visible above a water background; (7) specular reflections visible from the water surface; and (8) visual compatibility; ease of discriminating image detail. Recommendations for future recording over water bodies are included.

  14. The hamster polyomavirus--a brief review of recent knowledge.

    PubMed

    Scherneck, S; Ulrich, R; Feunteun, J

    2001-01-01

    The hamster polyomavirus (HaPV) was first described in 1967 as a virus associated with skin epithelioma of the Syrian hamster. The tumors appear spontaneously in a hamster colony bred in Berlin-Buch (HaB). Virus particles isolated from skin epitheliomas cause lymphoma and leukemia when injected into newborn hamsters from a distinct colony bred in Potsdam, Germany (HaP). The viral genome has been totally sequenced and the overall genetic organization establishes HaPV as a member of the polyomaviruses. HaPV is a second example of an middle T (MT) antigen encoding polyomavirus and nucleotide sequence homologies designates the mouse polyomavirus (Py) as the closest relative. Lymphomas induced by HaPV in HaP hamsters do not contain virus particles but instead accumulate different amounts of nonrandomly deleted free and/or integrated viral genomes. Transgenic mice produced by microinjection of HaPV DNA into the pronucleus of fertilized eggs of Gat: NMRI mice developed both, epitheliomas and lymphomas. Both tumor types contain extrachromosomal DNA. HaPV DNA was found to replicate in hamster lymphoid and fibroblast cell lines. Fully reproductive cycles could be detected only in GD36 lymphoblastic leukemia cells. HaPV carries the full transforming properties of a polyomavirus in vitro. Immortalization of primary rat cells is essentially carried out by the HaPV large T (LT) antigen and coexpression of HaPV MT and HaPV small T (ST) antigen is required for full transformation of rat fibroblasts. The preferential binding of HaPV MT to c-Fyn, a Src family kinase, has been proposed as a mechanism leading to lymphoid malignancies. Heterologous expression of HaPV-VP1 allowed the formation of virus like particles (VLPs) resembling HaPV particles. The high flexibility of HaPV-VP1 for insertion of foreign peptides offers a broad range of potential applications, especially in vaccine development. PMID:11210944

  15. Foodborne Transmission of Nipah Virus in Syrian Hamsters

    PubMed Central

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

    2014-01-01

    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 22C 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 5108 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 nave 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 and mitigating future outbreaks. PMID:24626480

  16. Integration Sites of Adenovirus Type 12 DNA in Transformed Hamster Cells and Hamster Tumor Cells

    PubMed Central

    Stabel, S.; Doerfler, W.; Friis, R. R.

    1980-01-01

    The patterns and sites of integration of adenovirus type 12 (Ad12) DNA were determined in three lines of Ad12-transformed hamster cells and in two lines of Ad12-induced hamster tumor cells. The results of a detailed analysis can be summarized as follows. (i) All cell lines investigated contained multiple copies (3 to 22 genome equivalents per cell in different lines) of the entire Ad12 genome. In addition, fragments of Ad12 DNA also persisted separately in non-stoichiometric amounts. (ii) All Ad12 DNA copies were integrated into cellular DNA. Free viral DNA molecules did not occur. The terminal regions of Ad12 DNA were linked to cellular DNA. The internal parts of the integrated viral genomes, and perhaps the entire viral genome, remained colinear with virion DNA. (iii) Except for line HA12/7, there were fewer sites of integration than Ad12 DNA molecules persisting. This finding suggested either that viral DNA was integrated at identical sites in repetitive DNA or, more likely, that one or a few viral DNA molecules were amplified upon integration together with the adjacent cellular DNA sequences, leading to a serial arrangement of viral DNA molecules separated by cellular DNA sequences. Likewise, in the Ad12-induced hamster tumor lines (CLAC1 and CLAC3), viral DNA was linked to repetitive cellular sequences. Serial arrangement of Ad12 DNA molecules in these lines was not likely. (iv) In general, true tandem integration with integrated viral DNA molecules directly abutting each other was not found. Instead, the data suggested that the integrated viral DNA molecules were separated by cellular or rearranged viral DNA sequences. (v) The results of hybridization experiments, in which a highly specific probe (143-base pair DNA fragment) derived from the termini of Ad12 DNA was used, were not consistent with models of integration involving true tandem integration of Ad12 DNA or covalent circularization of Ad12 DNA before insertion into the cellular genome. (vi) Evidence was presented that a small segment at the termini of the integrated Ad12 DNA in cell lines HA12/7, T637, and A2497-3 was repeated several times. The exact structures of these repeat units remained to be determined. The occurrence of these units might reflect the mechanism of amplification of viral and cellular sequences in transformed cell lines. Images PMID:7441821

  17. Altered somatosensory receptive fields in hamster colliculus after infraorbital nerve section and xylocaine injection.

    PubMed Central

    Jacquin, M F; Mooney, R D; Rhoades, R W

    1984-01-01

    The effects of acute infraorbital (i.o.) nerve section upon the responses of somatosensory cells in the rostral part of the deep layers of the hamster's superior colliculus were studied using standard extracellular single-unit recording and receptive field mapping techniques. In nine animals a given cell's receptive field was determined both before and after i.o. nerve section and, in all cases, new areas of sensitivity were unmasked within 15 min after the nerve was cut. In a given electrode penetration where the i.o. nerve was sectioned (n = 13), somatosensory cells recorded after the nerve was cut, as the electrode was being withdrawn from the colliculus, exhibited receptive fields considerably different from those of somatosensory cells isolated during the descent of the recording electrode. Seventeen deep-layer somatosensory cells (in eight hamsters) were tested before and after subcutaneous injections of xylocaine into their receptive fields. This manipulation unmasked new areas of cutaneous sensitivity for sixteen units. Of these, the new receptive fields of nine cells disappeared as sensitivity in the original receptive field returned; five ultimately retained both the new and old receptive fields; in two instances, sensitivity in the original receptive field never returned over the 3 h of testing. Control experiments (n = 7) demonstrated that the changes observed did not result from spontaneous alterations in receptive field borders, changes induced by variations in the level of general anaesthesia, or non-specific trauma associated with the xylocaine injections or the surgery required to expose the i.o. nerve. Images Fig. 7 Fig. 8 Plate 1 PMID:6716292

  18. High-throughput electrophysiology with Xenopus oocytes

    PubMed Central

    Papke, Roger L.; Smith-Maxwell, Cathy

    2010-01-01

    Voltage-clamp techniques are typically used to study the plasma membrane proteins, such as ion channels and transporters that control bioelectrical signals. Many of these proteins have been cloned and can now be studied as potential targets for drug development. The two approaches most commonly used for heterologous expression of cloned ion channels and transporters involve either transfection of the genes into small cells grown in tissue culture or the injection of the genetic material into larger cells. The standard large cells used for the expression of cloned cDNA or synthetic RNA are the egg progenitor cells (oocytes) of the African frog, Xenopus laevis. Until recently, cellular electrophysiology was performed manually, one cell at a time by a single operator. However, methods of high-throughput electrophysiology have been developed which are automated and permit data acquisition and analysis from multiple cells in parallel. These methods are breaking a bottleneck in drug discovery, useful in some cases for primary screening as well as for thorough characterization of new drugs. Increasing throughput of high-quality functional data greatly augments the efficiency of academic research and pharmaceutical drug development. Some examples of studies that benefit most from high-throughput electrophysiology include pharmaceutical screening of targeted compound libraries, secondary screening of identified compounds for subtype selectivity, screening mutants of ligand-gated channels for changes in receptor function, scanning mutagenesis of protein segments, and mutant-cycle analysis. We describe here the main features and potential applications of OpusXpress, an efficient commercially available system for automated recording from Xenopus oocytes. We show some types of data that have been gathered by this system and review realized and potential applications. PMID:19149490

  19. 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.

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

    PubMed

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

    2014-12-01

    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

  1. 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

  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. 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.

  4. 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

  5. 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.

  6. Active diffusion positions the nucleus in mouse oocytes.

    PubMed

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

    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. PMID:25774831

  7. 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...

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

    PubMed Central

    Castillo, G; Vera, J C; Yang, C P; Horwitz, S B; Rosen, O M

    1990-01-01

    The development of multidrug resistance (MDR) is associated with the overproduction of a plasma membrane glycoprotein, P glycoprotein. Here we 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 [3H]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. Images PMID:1693776

  9. Photodynamic therapy of hamster Greene melanoma in vitro and in vivo using bacteriochlorin-a as photosensitizer

    NASA Astrophysics Data System (ADS)

    Schuitmaker, J. J.; Van Best, Jaap A.; van Delft, J. L.; Jannink, J. E.; Oosterhuis, J. A.; Vrensen, Gijs F.; Ms Wolff-Rouendaal, Didi; Dubbelman, T. M.

    1996-01-01

    Efficient photodynamic therapy (PDT) of malignant melanoma may be possible with photosensitizers having absorption maxima in the far-red region e.g., above 700 nm. Bacteriochlorin a (BCA), a non toxic derivative of bacteriochlorphyllin a, has a high molecular absorption coefficient (32.000 M-1.cm-1) at 760 nm. At this wavelength tissue penetration of light is almost optimal and melanin absorption is relatively low. In several series of experiments BCA was proven to be a very effective photosensitizer, in vitro and in vivo. It is preferentially retained in experimental hamster Greene melanoma, rhabdomyosarcoma, RIF- and mamma tumors. Its fluorescence can be detected in vivo, thus enabling early tumor detection and it is rapidly cleared from the tissues which promises no, or minor skin photosensitivity. The effects of BCA-PDT were studied in vitro and in vivo using the heavily pigmented Hamster Greene Melanoma (HGM) cell line as a model. In vitro it was found that the uptake of BCA was time, concentration and temperature dependant. Upon illumination (10 Mw/cm2, 756 nm) after incubation with 2.5 (mu) g/ml BCA for 1 h, almost complete cell kill was obtained within seconds. Hamster Greene Melanoma implanted in the anterior eye chamber of rabbits is an accepted in vivo model for ocular melanoma. The effects of BCA-PDT using this model were studied by light- and electron microscopy. Immediately after PDT intracellular spaces were enlarged and blood vessels were clotted with swollen erythrocytes. Electron microscopy showed fused inner and outer membranes and affected cristae mitchondriales of some mitochondria. With time, the severity of tissue and cell damage increased. One day after irradiation tumor growth had stopped; fluorescein angiography showed non perfusion of the tumor. Histopathology showed almost complete tumor necrosis with occasionally viable cells at the tumor periphery. It is concluded that the direct mitochondrial damage and the vascular damage both contribute to BCA-PDT induced tumor necrosis.

  10. Natriuretic peptides stimulate oocyte meiotic resumption in bovine.

    PubMed

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

    2015-08-01

    The aim of the present study was to evaluate the expression of mRNA encoding natriuretic peptides (NPs) and their receptors in the cumulus-oocyte complex in cattle, a monovular mammalian species, and also to investigate the role of NPs in oocyte meiotic resumption in vitro. mRNA was observed for the NP precursor type-A (NPPA), type-C (NPPC), NP receptor-1 (NPR-1), receptor-2 (NPR-2) and receptor-3 (NPR-3) in bovine cumulus cells, and NPR-2 mRNA was observed in oocytes. These results are different from those obtained in mouse and pig models. The effects of NPPA, NP precursor type-B (NPPB) and NPPC on the resumption of arrested meiosis maintained by forskolin were studied at three different doses (10, 100 and 1000nM) with a 12h culture system. The germinal vesicle breakdown rates were greater (P?0.05) in oocytes that were cultured in the presence of one or a combination of NPs (from 44% to 73%) than the negative control (from 24% to 27%). Additionally, it was demonstrated that the concentration of cyclic guanosine 3',5'-monophosphate (cGMP) is increased by NPPA and NPPC in oocytes and cumulus cells after 3h of in vitro maturation. However, in both groups, the concentration of cyclic adenosine 3',5'-monophosphate (cAMP) in the oocyte did not increase between 3 and 6h of culture, even when forskolin was used. In summary, we observed the presence of mRNA for NPs and their receptors in the bovine cumulus-oocyte complex and demonstrated that, in vitro, NPPA, NPPB and NPPC stimulate oocyte meiotic resumption in a monovular species. PMID:26051611

  11. C-type natriuretic peptide inhibits porcine oocyte meiotic resumption.

    PubMed

    Hiradate, Yuki; Hoshino, Yumi; Tanemura, Kentaro; Sato, Eimei

    2014-08-01

    C-type natriuretic peptide (CNP) is a recently identified meiotic inhibitor in mice. However, it has not been investigated in porcine oocytes to date. This study aimed to demonstrate the inhibitory effect of CNP against germinal vesicle breakdown (GVBD) in porcine oocyte meiotic resumption. Immunohistochemical analysis revealed intense natriuretic peptide receptor 2 (NPR2) immunoreactivity in the oocyte surrounded cumulus cells in the follicles. Furthermore, reverse transcription polymerase chain reaction (RT-PCR) analysis showed the expression of npr2 mRNA only in cumulus cells but not in oocytes, suggesting that cumulus cells are the targets of CNP. When cumulus-oocyte complexes (COCs) or denuded oocytes (DOs) were cultured with various concentrations of CNP (10, 50, 100, 500, and 1,000 nM), inhibitory effect was observed in the COC group, but not in the DO group, confirming that CNP indirectly inhibits GVBD via cumulus cells. This evidence is the first indication that the CNP-NPR2 pathway is involved in meiotic arrest in porcine oocytes. Furthermore, we investigated the effect of oocyte-derived paracrine factor (ODPF) on npr2 mRNA expression level in cumulus cells by evaluating changes in mRNA expression in oocytectomised COCs (OXCs) by real-time PCR. A significant decrease in npr2 mRNA expression level was observed in OXCs, whereas mRNA expression level was restored in OXCs with DOs, indicating that ODPF participates in the regulation of npr2 expression in porcine cumulus cells. PMID:23331536

  12. Visualization of mRNA Localization in Xenopus Oocytes

    PubMed Central

    Gagnon, James A.; Mowry, Kimberly L.

    2011-01-01

    Visualization of in vivo mRNA localization provides a tool for understanding steps in the mechanism of transport. Here we detail a method of fluorescently labeling mRNA transcripts and microinjecting them into Xenopus laevis oocytes followed with imaging by confocal microscopy. This technique overcomes a significant hurdle of imaging RNA in the frog oocyte while providing a rapid method of visualizing mRNA localization in high resolution. PMID:21431735

  13. In vitro fertilization and subsequent development of porcine oocytes using cryopreserved and liquid-stored spermatozoa from various boars.

    PubMed

    Suzuki, Chie; Yoshioka, Koji; Itoh, Seigo; Kawarasaki, Tatsuo; Kikuchi, Kazuhiro

    2005-10-01

    We had previously developed a porcine IVF system using a chemically defined medium, i.e., porcine gamete medium supplemented with theophylline, adenosine, and cysteine (PGMtac). In the present study, we investigated the utility of this IVF system using different types of semen: (1) cryopreserved ejaculated (n = 8); (2) cryopreserved epididymal (n = 4); and (3) liquid-stored ejaculated (n = 5). Cryopreserved spermatozoa were prepared by three methods. In vitro-matured porcine oocytes were fertilized for 20 h in PGMtac using each type of semen, and the presumptive zygotes were cultured in porcine zygote medium (PZM)-4 for 5 days. In the case of frozen-thawed spermatozoa, the number of spermatozoa per penetrated oocyte (1.1-1.7), rate of blastocyst formation (26-56%), and total number of cells per blastocyst (34-49) differed (P < 0.05) among freezing methods. However, blastocysts were produced using all types of cryopreserved spermatozoa (14-75%). When spermatozoa were liquid-stored for 1-14 days after semen collection, the rate of sperm penetration (P < 0.05) decreased as storage time increased, although there was no significant reduction in sperm motility during storage. In all groups, semen that had been stored within 10 days after collection enabled blastocyst production in vitro (20-48%). In conclusion, this IVF system, which uses a chemically defined medium, had widespread utility with both frozen-thawed and liquid-stored spermatozoa. PMID:16139605

  14. Efficient Gene Targeting in Golden Syrian Hamsters by the CRISPR/Cas9 System

    PubMed Central

    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. PMID:25299451

  15. ROCK inhibitor Y-27632 prevents porcine oocyte maturation.

    PubMed

    Zhang, Yu; Duan, Xing; Xiong, Bo; Cui, Xiang-Shun; Kim, Nam-Hyung; Rui, Rong; Sun, Shao-Chen

    2014-07-01

    The inhibitor Y-27632 is a specific selective inhibitor of Rho-associated protein kinases (ROCKs), which are downstream effectors of Rho guanosine triphosphatease (GTPases) and regulate Rho-associated cellular functions, including actin cytoskeletal organization. Little is known regarding the effects of Y-27632 on mammalian oocyte maturation. In the present study, we investigated the effects of Y-27632 on porcine oocyte meiosis and possible regulatory mechanisms of ROCK during porcine oocyte maturation. We found that ROCK accumulated not only at spindles, but also at the cortex in porcine oocytes. Y-27632 treatment reduced ROCK expression, and inhibited porcine oocyte meiotic maturation, which might be because of the impairment of actin expression and actin-related spindle positioning. Y-27632 treatment also disrupted the formation of actin cap and cortical granule-free domain, which further confirmed a spindle positioning failure. Thus, Y-27632 has significant effects on the meiotic competence of mammalian oocytes by reducing ROCK expression, and the regulation is related to its effects on actin-mediated spindle positioning. PMID:24681214

  16. 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, Ada; 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 (2545 years of age) and 24 IVF oocyte donors (1833 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

  17. Non-meiotic chromosome instability in human immature oocytes.

    PubMed

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

    2014-02-01

    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

  18. The Role of Mitochondria from Mature Oocyte to Viable Blastocyst

    PubMed Central

    2013-01-01

    The oocyte requires a vast supply of energy after fertilization to support critical events such as spindle formation, chromatid separation, and cell division. Until blastocyst implantation, the developing zygote is dependent on the existing pool of mitochondria. That pool size within each cell decreases with each cell division. Mitochondria obtained from oocytes of women of advanced reproductive age harbor DNA deletions and nucleotide variations that impair function. The combination of lower number and increased frequency of mutations and deletions may result in inadequate mitochondrial activity necessary for continued embryo development and cause pregnancy failure. Previous reports suggested that mitochondrial activity within oocytes may be supplemented by donor cytoplasmic transfer at the time of intracytoplasmic sperm injection (ICSI). Those reports showed success; however, safety concerns arose due to the potential of two distinct populations of mitochondrial genomes in the offspring. Mitochondrial augmentation of oocytes is now reconsidered in light of our current understanding of mitochondrial function and the publication of a number of animal studies. With a better understanding of the role of this organelle in oocytes immediately after fertilization, blastocyst and offspring, mitochondrial augmentation may be reconsidered as a method to improve oocyte quality. PMID:23766762

  19. Genetic analysis of the oocyte--a review.

    PubMed

    Plachot, M

    2003-10-01

    Chromosome number abnormalities are remarkably common in human reproduction. Most are caused by chromosomal non-disjunction and premature chromatid separation in oocyte meiosis I. Pooled data from previous studies showed that one in five oocytes that failed to fertilize after in vitro insemination was abnormal when analysed by conventional cytogenetics. Preconception genetic diagnosis, carried out on the first and second polar bodies by FISH, using 5 chromosome-specific probes (13, 16, 18, 21 and 22), showed that the rate of aneuploidy is higher in women aged 35 or over (52.1 per cent). Oocyte dysmorphy seems to have little effect on the rate of aneuploidy except for giant oocytes, which are usually diploid and may cause triploidy after fertilization. Intra- and extrafollicular influences (perifollicular microvasculature, oxygenation, the presence of residues from cigarette smoke) may disturb maturation, leading to immaturity and aneuploidy. Thus, oocyte meiosis is very sensitive to endogenous and exogenous factors that may cause the production of oocytes with chromosomal abnormalities and therefore, of abnormal zygotes. PMID:14559033

  20. 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.

  1. 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.

  2. Human oocytes. Error-prone chromosome-mediated spindle assembly favors chromosome segregation defects in human oocytes.

    PubMed

    Holubcov, Zuzana; Blayney, Martyn; Elder, Kay; Schuh, Melina

    2015-06-01

    Aneuploidy in human eggs is the leading cause of pregnancy loss and several genetic disorders such as Down syndrome. Most aneuploidy results from chromosome segregation errors during the meiotic divisions of an oocyte, the egg's progenitor cell. The basis for particularly error-prone chromosome segregation in human oocytes is not known. We analyzed meiosis in more than 100 live human oocytes and identified an error-prone chromosome-mediated spindle assembly mechanism as a major contributor to chromosome segregation defects. Human oocytes assembled a meiotic spindle independently of either centrosomes or other microtubule organizing centers. Instead, spindle assembly was mediated by chromosomes and the small guanosine triphosphatase Ran in a process requiring ~16 hours. This unusually long spindle assembly period was marked by intrinsic spindle instability and abnormal kinetochore-microtubule attachments, which favor chromosome segregation errors and provide a possible explanation for high rates of aneuploidy in human eggs. PMID:26045437

  3. Sidewall penetrator for oil wells

    NASA Technical Reports Server (NTRS)

    Collins, E. R., Jr.

    1981-01-01

    Penetrator bores horizontal holes in well casing to increase trapped oil drainage. Several penetrators operated by common drive are inserted into well at once. Shaft, made from spiraling cable, rotates and thrusts simultaneously through rigid curvilinear guide tube forcing bit through casing into strata. Device pierces more deeply than armor-piercing bullets and shaped explosive charges.

  4. Ground-penetrating radar methods

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Ground-penetrating radar geophysical methods are finding greater and greater use in agriculture. With the ground-penetrating radar (GPR) method, an electromagnetic radio energy (radar) pulse is directed into the subsurface, followed by measurement of the elapsed time taken by the radar signal as it ...

  5. DOSE RESPONSE OF ELASTASE-INDUCED EMPHYSEMA IN HAMSTERS

    EPA Science Inventory

    Elastase-induced emhysema 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. It appears that as little as 6 units of elastase produces mild emphysema in hamste...

  6. CARCINOGENIC POTENTIAL OF ROTENONE. PHASE I: DIETARY ADMINISTRATION TO HAMSTERS

    EPA Science Inventory

    Studies were performed to evaluate the potential carcinogenicity rotenone in the Syrian Golden hamster. Several ancillary range-finding studies were carried out including 14-day feeding trials and a reproduction experiment. The latter experiment indicated that rotenone at a level...

  7. PULMONARY CELL POPULATIONS IN HAMSTERS MAINTAINED UNDER EGYPTIAN LABORATORY CONDITIONS

    EPA Science Inventory

    The study was conducted to obtain baseline values for pulmonary cells in golden hamsters (Mesocricetus auratus) bred and maintained under the laboratory conditions of Al-Azhar University in Egypt. An improvised technique is presented for measuring pulmonary cells obtained by lung...

  8. Development of Taenia pisiformis in golden hamster (Mesocricetus auratus)

    PubMed Central

    2011-01-01

    The life cycle of Taenia pisiformis includes canines as definitive hosts and rabbits as intermediate hosts. Golden hamster (Mesocricetus auratus) is a rodent that has been successfully used as experimental model of Taenia solium taeniosis. In the present study we describe the course of T. pisiformis infection in experimentally infected golden hamsters. Ten females, treated with methyl-prednisolone acetate were infected with three T. pisiformis cysticerci each one excised from one rabbit. Proglottids released in faeces and adults recovered during necropsy showed that all animals were infected. Eggs obtained from the hamsters' tapeworms, were assessed for viability using trypan blue or propidium iodide stains. Afterwards, some rabbits were inoculated with eggs, necropsy was performed after seven weeks and viable cysticerci were obtained. Our results demonstrate that the experimental model of adult Taenia pisiformis in golden hamster can replace the use of canines in order to study this parasite and to provide eggs and adult tapeworms to be used in different types of experiments. PMID:21787386

  9. Development of Taenia pisiformis in golden hamster (Mesocricetus auratus).

    PubMed

    Toral-Bastida, Elizabeth; Garza-Rodriguez, Adriana; Jimenez-Gonzalez, Diego E; Garcia-Cortes, Ramon; Avila-Ramirez, Guillermina; Maravilla, Pablo; Flisser, Ana

    2011-01-01

    The life cycle of Taenia pisiformis includes canines as definitive hosts and rabbits as intermediate hosts. Golden hamster (Mesocricetus auratus) is a rodent that has been successfully used as experimental model of Taenia solium taeniosis. In the present study we describe the course of T. pisiformis infection in experimentally infected golden hamsters. Ten females, treated with methyl-prednisolone acetate were infected with three T. pisiformis cysticerci each one excised from one rabbit. Proglottids released in faeces and adults recovered during necropsy showed that all animals were infected. Eggs obtained from the hamsters' tapeworms, were assessed for viability using trypan blue or propidium iodide stains. Afterwards, some rabbits were inoculated with eggs, necropsy was performed after seven weeks and viable cysticerci were obtained. Our results demonstrate that the experimental model of adult Taenia pisiformis in golden hamster can replace the use of canines in order to study this parasite and to provide eggs and adult tapeworms to be used in different types of experiments. PMID:21787386

  10. Parameters of Mycoplasma pneumoniae infection in Syrian hamsters.

    PubMed Central

    Barile, M F; Chandler, D K; Yoshida, H; Grabowski, M W; Harasawa, R; Razin, S

    1988-01-01

    An animal model for evaluating the potency of Mycoplasma pneumoniae vaccines was developed with hamsters. Factors that influence hamster infection by M. pneumoniae were defined, and parameters for assessment of intensity of pulmonary disease were established. Colonization of hamster lungs was determined by culture, and intensity of lung disease was assessed histopathologically and expressed numerically as a lung pathological score. Intratracheal inoculation of the challenge was superior to the intranasal or aerosol route for inducing a consistent degree of lung disease. A challenge dose of 10(6) CFU inoculated intratracheally produced lung colonization and significant reproducible lung pathological scores in essentially all unvaccinated animals. The peak of infection, as determined by these criteria, was at about 2 weeks after challenge. Animals over 6 weeks of age were preferable for the test, since younger animals exhibited a lower lung pathological score even though they showed the same degree of lung colonization. The hamster assay developed provides a dependable experimental system for testing the protective potency of M. pneumoniae vaccines. Images PMID:3137169

  11. RELATIONSHIP BETWEEN AUTONOMIC AND BEHAVIORAL THERMOREGULATION IN THE GOLDEN HAMSTER

    EPA Science Inventory

    Preferred ambient temperature (Ta) of male golden hamsters (Mesocricitus auratus) was measured repeatedly by placing the animals in a temperature gradient for 80 min. A total of 180 observations were made during the last 20 min of treatment in the gradient. The mean preferred Ta ...

  12. 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. PMID:25499542

  13. Cytogenetic analysis of horse oocytes matured in vitro for different periods of time.

    PubMed

    Sosnowski, J; Lechniak, D; Brzozowska, M; Swito?ski, M

    1997-01-01

    This paper presents the results of recovering horse oocytes by aspiration and maturation in vitro for 24, 30, 36 or 42 h. A total of 522 oocytes were recovered from 221 ovaries (2.4 per ovary) and 271 oocytes (51.9%) were selected for in vitro maturation (IVM). Oocytes were cultured in maturation medium (TCM 199 + estrus cow serum [ECS] + follicle-stimulating hormone [FSH] + 17 beta-estradiol + gentamycin). One hundred and seventy oocytes were cytogenetically analysed (68.3%). Cytogenetic analysis showed that the stage of maturation (first telophase-TI or second metaphase-MII) for fertilization was achieved in 42.2% of oocytes after 24 h, 70.4% of oocytes after 30 h, 75.0% of oocytes after 36 h and 74.4% of oocytes after 42 h of in vitro maturation. PMID:9115597

  14. 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. PMID:26278808

  15. Energy Status Characteristics of Porcine Oocytes During In Vitro Maturation is Influenced by Their Meiotic Competence.

    PubMed

    Milakovic, I; Jeseta, M; Hanulakova, S; Knitlova, D; Hanzalova, K; Hulinska, P; Machal, L; Kempisty, B; Antosik, P; Machatkova, M

    2015-10-01

    The characteristics of energy status in porcine oocytes as related to their meiotic competence and in vitro maturation were studied. Cycling pubertal gilts in the early luteal to early follicular phases of the ovarian cycle were used as oocyte donors. The oocytes recovered from medium (MF) or small follicles (SF) were considered meiotically more or less competent, respectively. A half of oocytes from each category was matured by the standard protocol. The oocytes were examined before or after maturation by confocal microscopy, a bioluminescent cell assay and Western blotting. Four experiments, each in triplicate, were performed to assess both SF and MF oocytes in terms of metabolic units formed by mitochondria and lipids, ATP and lipid consumption and lipid droplets with adipose differentiation-related protein (ADRP) expression. The proportion of oocytes with metabolic units, the mean ATP content and the number of lipid droplets per oocyte, and the relative number of lipid droplets with ADRP expression were significantly higher in the MF compared to SF oocytes before maturation. On the other hand, after maturation, there was an increase in the proportion of oocytes with metabolic units and the relative number of lipid droplets with ADRP expression in the SF compared to MF oocytes. In conclusion, specific differences in energy characteristics between porcine oocytes with different meiotic competence were found. Meiotically more competent oocytes are more advanced in terms of energy reserves before maturation, while meiotically less competent oocytes are more active in replenishing energy stores during maturation. PMID:26280917

  16. Oocyte morphology does not affect fertilization rate, embryo quality and implantation rate after intracytoplasmic sperm injection.

    PubMed

    Balaban, B; Urman, B; Sertac, A; Alatas, C; Aksoy, S; Mercan, R

    1998-12-01

    In this study, we compared the fertilization rate and embryo quality after intracytoplasmic sperm injection (ICSI) as they relate to oocyte morphology. A total of 654 ICSI cycles yielding 5903 metaphase II oocytes were observed. The oocytes retrieved in these cycles were divided into (i) normal oocytes, (ii) oocytes with extracytoplasmic abnormalities (dark zona pellucida and large perivitelline space), (iii) oocytes with cytoplasmic abnormalities (dark cytoplasm, granular cytoplasm, and refractile body), (iv) oocytes with shape abnormalities, and (v) oocytes with more than one abnormality (double and triple abnormalities). Intracytoplasmic vacuoles and aggregates of smooth endoplasmic reticulum were not recorded separately. The fertilization rate and quality of morphologically graded embryos did not differ between the groups. There were 77 cycles where all transferred embryos were derived from abnormal oocytes, and 164 cycles where all embryos were derived from normal oocytes. These cycles were studied further. The two groups were comparable regarding mean female age, duration of infertility, duration of ovarian stimulation, number of ampoules of gonadotrophin injected, and number of oocytes retrieved. Two clinical pregnancy rates (44.4 versus 42.1%) and implantation rates per embryo (10.3 versus 13.2%) were similar. In conclusion, in couples undergoing ICSI, abnormal oocyte morphology is not associated with a decreased fertilization rate or unfavourable embryo quality. Furthermore, embryos derived from abnormal oocytes yield similar clinical pregnancy and implantation rates when transferred compared with embryos derived from normal oocytes. PMID:9886529

  17. 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.

  18. Differential osmotic behavior of mammalian oocytes before and after maturation: a quantitative analysis using goat oocytes as a model.

    PubMed

    Le Gal, F; Gasqui, P; Renard, J P

    1994-04-01

    This study shows that immature and mature goat oocytes respond differently to hyperosmotic stress; when exposed to a 1.5 M propanediol solution, immature oocytes manifest a higher osmotic stress than do mature oocytes. This is the consequence of both higher water permeability (133.9 +/- 15.2 vs 82.4 +/- 4.4 x 10(-3) cm/min) and lower propanediol permeability (0.87 +/- 0.03 vs 1.20 +/- 0.03 x 10(-3) cm/min at 20 degrees C) in the immature than in the mature stage. The difference of osmotic behavior between these two types of oocytes is abolished following exposure to cytochalasin D, a drug known to modify the cellular microfilament network. This result suggests differences in actin organization between the two types of oocyte, probably at the cortical level. Calculated values of the intracellular concentration of propanediol as a function of time of exposure show that propanediol rapidly permeates both types of oocyte and that the kinetics of intracellular concentration are lowered by cytochalasin treatment. PMID:8004996

  19. Blood clots in the cumulus-oocyte complex predict poor oocyte quality and post-fertilization development.

    PubMed

    Ebner, T; Moser, M; Shebl, O; Sommergruber, M; Yaman, C; Tews, G

    2008-06-01

    Assessment of oocyte maturity and quality (morphological appearance) at the time of retrieval is difficult as the egg is obscured by a large cumulus mass that hinders adequate scoring. Since no data are available on the possible relationship between the cumulus-oocyte complex (COC) and oocyte morphology, this prospective intracytoplasmic sperm injection study was set up in 87 consecutive patients. COC were grouped according to expansion of both corona radiata and cumulus matrix. Special emphasis was placed on recording morphological anomalies of COC (inclusion of blood clots and amorphous clumps). For all mature ovae, quality was assessed and preimplantation development followed up to blastocyst stage if fertilized. The risk of not harvesting an oocyte was higher in COC with blood clots compared with normal cumulus matrices (P = 0.004). COC expansion did not allow for prediction of either nuclear status or quality of the egg. The presence of blood clots within the cumulus matrix was associated with reduced oocyte quality (dense central granulation), fertilization rate and blastocyst formation, compared with unaffected COC (P < 0.05). It may be postulated that COC showing blood inclusions derive from poor quality follicles, which has a detrimental effect on oocyte quality and further cleavage to blastocyst stage. Consequently, mechanical removal of blood clots cannot rescue the corresponding embryo. PMID:18549689

  20. 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 (0.754.0 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

  1. Anti-Mllerian Hormone (AMH), Inhibin-?, Growth Differentiation Factor 9 (GDF-9), and Bone Morphogenic Protein-15 (BMP-15) mRNA and protein are influenced by photoperiod-induced ovarian regression and recrudescence in Siberian hamster ovaries

    PubMed Central

    Shahed, Asha; Young, Kelly A.

    2013-01-01

    Exposure of Siberian hamsters to short photoperiod (SD) inhibits ovarian function, including folliculogenesis, whereas function is restored with transfer to long photoperiods (LD). To investigate the mechanism of photostimulated recrudescence, we assessed key folliculogenic factors: anti-Mllerian hormone (AMH), inhibin-?, growth differentiation factor-9 (GDF-9), and bone morphogenic protein-15 (BMP-15) across the estrus cycle and in photoregressed and recrudescing ovaries. Adult hamsters were exposed to either LD or SD for 14 weeks representing functional and regressed ovaries, respectively. Select regressed hamsters were transferred back to LD for two (post-transfer week 2; PTw2) or eight weeks (PTw8). Ovaries were collected and fixed in formalin for immunohistochemistry or frozen in liquid nitrogen for real-time PCR. AMH, inhibin-?, GDF-9 and BMP-15 mRNA and protein were detected in all stages of the estrus cycle. Fourteen weeks of SD exposure increased (p<0.05) ovarian AMH, GDF-9 and BMP-15, but not inhibin-? mRNA levels as compared to LD. Transfer of regressed hamsters to stimulatory long photoperiod for 8 weeks returned AMH and GDF-9 mRNA levels to LD levels levels, and further increased mRNA levels for inhibin-? and BMP-15. Immunostaining of AMH, inhibin-?, GDF-9 and BMP-15 proteins was most intense in preantral/antral follicles and oocytes. The overall immunostaining extent for AMH and inhibin-? generally mirrored mRNA data, though no changes were observed in GDF-9 or BMP-15 immunostaining extent. Shifts in mRNA and protein levels across photoperiod conditions suggest possible syncretic roles for these folliculogenic factors in photostimulated recrudescence via potential regulation of follicle recruitment, preservation and development. PMID:23877969

  2. Cloning and characterization of SOB1, a new testis-specific cDNA encoding a human sperm protein probably involved in oocyte recognition.

    PubMed

    Lefevre, A; Duquenne, C; Rousseau-Merck, M F; Rogier, E; Finaz, C

    1999-05-27

    A human sperm-oocyte binding protein, SOB1, was purified by two dimensional gel electrophoresis and sequenced. This protein was selected because it was recognized by a monoclonal antibody that inhibited the binding of human sperm to zona-free hamster oocytes. The sequences of the tryptic peptides were used to design degenerate primers. These were used to amplify a specific fragment from human testis cDNA by the polymerase chain reaction. This 1233 bp fragment was extended in 3' and 5' by RACE to obtain the 3 kb full length SOB1 cDNA. Sequence analysis indicated that the deduced open reading frame encodes a 853 amino acid protein, with a molecular mass of 94. 7 kDa. This is a new testis-specific cDNA. It is 27, 32.8 and 34.4% homologous to three sperm proteins, HI, Fsc1 and AKAP82 respectively. A single 3kb transcript was demonstrated only in the testis by northern blot analysis. It is a single copy gene, well conserved among mammals and located on human chromosome 12 at band p13. PMID:10334916

  3. Cooperative transmembrane penetration of nanoparticles.

    PubMed

    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

  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. PMID:24184862

  5. 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

  6. Genome organization and epigenetic marks in mouse germinal vesicle oocytes.

    PubMed

    Bonnet-Garnier, Amelie; Feuerstein, Prisca; Chebrout, Martine; Fleurot, Renaud; Jan, Habib-Ullah; Debey, Pascale; Beaujean, Nathalie

    2012-01-01

    During the final step of oogenesis, the oocyte nucleus is subject to large-scale modifications that correlate with transcriptional silencing. While oocytes with dense chromatin around the nucleolus are silent (SN, surrounded nucleolus), oocytes with uncondensed chromatin (NSN, non-surrounded nucleolus) are transcriptionally active. It is believed that epigenetic mechanisms that participate in gene expression regulation could play a role in this event. In this context, we examined the behaviour of heterochromatin and related histone modifications during the NSN to SN transition by immunostaining. Using fluorescent in situ hybridization on three dimensional-preserved nuclei (3D-FISH), we also studied the distribution of centromeric, pericentromeric and ribosomal (rDNA) sequences in relation to the nucleolus (also called the nucleolus-like body, NLB). We observed that in NSN-type oocytes, pericentromeric heterochromatin is aggregated within chromocenters. In SN-type oocytes, pericentromeric heterochromatin and centromeres form a discontinuous ring around the NLB. rDNA sequences, which initially present a pearl necklace structure, gather together in seven highly condensed foci at the NLB periphery. H3K9me3 and H4K20me3 heterochromatin marks clearly label chromocenters, whereas H3K4me3 and H4K5ac are totally excluded from heterochromatin regions, even in the very compact SN-nuclei. Remarkably, H3K27me3 displays an intermediate behavior. It appears that GV oocyte nuclei exhibit a specific epigenetic landscape. Histone modifications, related to both active and repressive chromatin structures, seem to follow the large-scale chromatin movements that occur during the NSN to SN transition. We also demonstrate that, while heterochromatin regions re-localize around the NLB, rDNA sequences adopt a highly compact structure in SN-type oocytes. PMID:23417410

  7. 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 ?M. 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 ?M, 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. PMID:24047285

  8. 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

  9. Successful pregnancy and delivery after ICSI with artificial oocyte activation by calcium ionophore in in-vitro matured oocytes: a case report.

    PubMed

    Kim, Jun-Woo; Yang, Seong-Ho; Yoon, San-Hyun; Kim, Sang-Don; Jung, Jae-Hoon; Lim, Jin-Ho

    2015-04-01

    The achievement of a successful pregnancy and delivery after oocyte activation with calcium ionophore is reported in a couple having low fertilization rates after intracytoplasmic sperm injection (ICSI) of in-vitro matured oocytes. A couple, in which the wife had polycystic ovary syndrome and the husband had moderate oligoteratozoospermia, showed a low fertilization rate in a previous in-vitro maturation cycle (2/11 [18.2%]). The most likely cause of complete fertilization failure or low fertilization rates is failure of oocyte activation. Therefore, artificial oocyte activation by calcium ionophore was combined with ICSI to achieve viable fertilized oocytes. Oocytes were stimulated with calcium ionophore for 30?min after ICSI. The fertilization rate of oocytes activated with calcium ionophore (13/15 [86.7%] and 7/9 [77.8%]) was higher than that of the non-activated oocytes. In the latest cycle, three embryos derived from the activated oocytes were transferred into the uterus on day 3. Subsequently, two gestational sacs were identified on ultrasound. The patient delivered dizygotic twins (girl 2260?g and boy 2760?g) at 35 weeks and 6 days gestation by caesarean section. This result suggests that calcium ionophore could be useful for oocyte fertilization in couples with low fertilization rates after ICSI of in-vitro matured oocytes. PMID:25592974

  10. Experimental investigation of wavelength dependence of penetration depth and imaging contrast for ultrahigh-resolution optical coherence tomography

    NASA Astrophysics Data System (ADS)

    Ishida, S.; Nishizawa, N.; Itoh, K.

    2011-03-01

    Optical coherence tomography (OCT) is a non invasive optical imaging technology for micron-scale cross-sectional imaging of biological tissue and materials. Although OCT has many advantages in medical equipments, low penetration depth is a serious limitation for other applications. To realize the ultrahigh resolution and the high penetration depth at the same time, it is effective to choose the proper wavelength to maximize the light penetration and enhance the image contrast at deeper depths. Recently, we have demonstrated ultrahigh resolution and high penetration depth OCT by use of all-fiber based Gaussian shaped supercontinuum source at 1.7 ?m center wavelength. Gaussian-like supercontinuum with 360 nm bandwidth at center wavelength of 1.7 ?m was generated by ultrashort pulse Er doped fiber laser based system. In this paper, using 0.8 ?m and 1.3 ?m SC sources in addition to the 1.7 ?m SC source, we have investigated the wavelength dependence of ultrahigh resolution OCT in terms of penetration depth. Longitudinal resolutions at each wavelength region are almost 4.6 ?m in air. The obtained sensitivity was 95 dB for all wavelength regions. We confirmed the difference of imaging contrast and penetration depth with hamster's cheek pouch and so on. As the wavelength was increased, the magnitude of penetration depth was increased for these samples.

  11. 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

  12. Modification of aortic contractility in the cardiomyopathic hamster.

    PubMed Central

    Dumont, E. C.; Lambert, C.; Lamontagne, D.

    1996-01-01

    1. The functional arterial response in the cardiomyopathic hamster compared with inbred control, was investigated in thoracic aortae. For this purpose, vessels were cut into 6-mm rings and mounted in 20-ml organ baths. 2. In a first experimental series, the function of the endothelium was evaluated. Dose-response curves to acetylcholine (0.1 nM-10 microM) on phenylephrine (0.3 microM)-preconstricted rings of cardiomyopathic hamsters and inbred age-matched controls were comparable (log[EC50] of -7.08 +/- 0.12 and -7.18 +/- 0.12, respectively; n = 4). 3. Changes in contractility of cardiomyopathic hamster endothelium-denuded aortae were investigated. Dose-response curves to phenylephrine (1 nM-0.1 mM), angiotensin II (10 pM-0.3 microM), 5-hydroxytryptamine (5-HT) (1 nM-0.1 mM) and KCl (1 mM-0.1 M) were performed. Increased sensitivity in cardiomyopathic hamster aortae, compared to controls, was observed with phenylephrine (log[EC50] of -7.25 +/- 0.05 and -6.83 +/- 0.05, respectively, n = 6, P < 0.001) and angiotensin II (log[EC50] of -8.67 +/- 0.07 and -8.26 +/- 0.06, respectively, n = 6, P = 0.001) but not with 5-HT or KCl. A decreased maximum response in cardiomyopathic, compared to control, was observed with 5-HT (1.28 +/- 0.06 g vs 1.56 +/- 0.07 g, respectively, n = 6, P = 0.03). Comparable results were found in aortae with an intact endothelium. 4. No difference in the maximum contractile response to the G-protein activator, NaF (3, 10 and 30 mM) was observed in either group of animals. 5. Phorbol 12-myristate 13-acetate (PMA, 1-10 microM) was used to assess changes in the activity of protein kinase C (PKC). Contractility to PMA was increased in cardiomyopathic hamster aortae compared to controls (0.22 +/- 0.02 g vs 0.07 +/- 0.03 g at 3 microM, respectively, n = 6, P = 0.003). 6. Finally, cardiomyopathic hamsters aortae were found to be less sensitive when exposed to increasing concentrations of Ca2+ (10 microM-1 mM) in KCl-depolarized rings (0.58 +/- 0.04 g in cardiomyopathic vs 0.79 +/- 0.06 g in control aortae at 0.3 mM, n = 8, P = 0.03). 7. In conclusion, aortae from cardiomyopathic hamsters are more sensitive to phenylephrine and angiotensin II, but not to 5-HT, than those of controls. The increase in sensitivity does not implicate Ca2+ channels or Ca2+ itself since cardiomyopathic hamsters aortae are not more sensitive to KCl- and Ca(2+)-induced contraction. The greater effect of PMA on cardiomyopathic hamster aortae suggests that the increase in sensitivity to phenylephrine and angiotensin II involves an enhanced activity of PKC. PMID:8818336

  13. 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. PMID:25245076

  14. In vitro developmental competence of bovine oocytes: Effect of corpus luteum and follicle size

    PubMed Central

    Karami Shabankareh, Hamed; Shahsavari, Mohammad Hamed; Hajarian, Hadi; Moghaddam, Gholamali

    2015-01-01

    Background: Previous studies reported many discrepancies about the effects of corpus luteum (CL) and ovarian follicle size on the developmental competence of oocytes. Objective: The aim of this study was to investigate the effects of CL and different size of follicle on the developmental potential of bovine oocytes. Materials and Methods: After ovarian classification based on presence or absence of CL, sample follicles were placed in three groups according to their diameter; small (S; 36 mm), medium (M; 69 mm), and large (L; 1020 mm). Collected oocytes in each group were subjected to the in vitro embryo production processes. Results: Results showed that, the percentages of blastocyst obtained from oocytes originating from small and medium follicles of ovaries bearing a CL (CL+S-oocytes and CL+M-oocytes, respectively) were lower (p<0.001) than those of small and medium follicles of ovaries not bearing a CL (CL-S-oocytes and CL-M-oocytes, respectively) (30.8% and 33.6% vs. 36.9% and 38.7% respectively). Although, the percentages of blastocyst obtained from CL-M-oocytes and CL-L-oocytes were greater (p< 0.001) than those of CL+S-oocytes and CL+M-oocytes. There were no significant differences in the percentages of blastocyst formation between controls (C-oocytes), CL-S-oocytes and CL+L-oocytes. Conclusion: According to the results of this study, the negative effect of CL on the developmental competence of bovine oocyte depends on the follicle size. Therefore, oocytes originating from large grown follicles were not influenced by negative effects of CL as much as those originating from small and medium follicles did. PMID:26644789

  15. KL/KIT co-expression in mouse fetal oocytes.

    PubMed

    Doneda, Luisa; Klinger, Francesca-Gioia; Larizza, Lidia; De Felici, Massimo

    2002-12-01

    The tyrosine kinase receptor, KIT, and its ligand, KL are important regulators of germ cell development. The aim of this study was to examine in detail the expression of the genes encoding these proteins (White and Steel, respectively) during the fetal period (14.5-18.5 days post coitum, dpc) and the two weeks after birth in mouse ovaries using the highly sensitive in situ reverse-transcriptase polymerase chain reaction (in situ RT-PCR). KL and KIT mRNAs were not detected in 14.5-15.5 dpc ovaries but, between 16.5 and 17.5 dpc, most of the oocytes in the outer regions of the ovaries positively stained for both mRNAs. The majority of the co-expressing oocytes were identified at the zygotene/pachytene stage of meiotic prophase I. At 18.5 dpc, positive staining for KL mRNA was present only in the somatic cells in the outer regions of the ovaries. At birth, faint KL mRNA-labelled somatic cells were mainly found in the central region of the ovaries and, by P7-14, a higher level of expression was detected in the follicle cells of one- and two-layered growing follicles. Between 17.5 dpc and birth, most of the oocytes expressed KIT mRNA and, from P7 onward, there was a considerable accumulation of transcripts in the growing oocytes. The results of in situ RT-PCR were confirmed by RT-PCR on purified populations of oocytes, and at protein level by means of immunohistochemistry. The co-expression of KL and KIT in a fraction of fetal oocytes suggests that the KL/KIT system, besides the well known paracrine functions on germ cells, may exert a novel autocrine role during the mid-stage of the oocyte meiotic prophase. The possibility that this autocrine loop plays a role in sustaining the survival of fetal oocytes in this stage is supported by the finding that the addition to the culture medium of anti-KL or anti-KIT antibodies led to a significant increase in oocyte apoptosis in the absence of exogenous KL. PMID:12533025

  16. Naturally occurring mastitis disrupts developmental competence of bovine oocytes.

    PubMed

    Roth, Z; Dvir, A; Kalo, D; Lavon, Y; Krifucks, O; Wolfenson, D; Leitner, G

    2013-10-01

    We examined the effects of naturally occurring mastitis on bovine oocyte developmental competence in vitro. Specifically, we investigated the effects of intramammary infection on the ovarian pool of oocytes (i.e., follicle-enclosed oocytes) and their ability to undergo in vitro maturation, fertilization, and further development to the blastocyst stage. Culled Holstein cows (n=50) from 9 commercial dairy farms in Israel were allotted to 3 groups according to somatic cell count (SCC) records of the last 3 monthly milk tests as well as of quarter samples collected before slaughter: (1) low SCC (n=7), (2) medium SCC (n=16), or (3) high SCC (n=27). Means of SCC values differed among low-, medium-, and high-SCC groups: 148,000, 311,000 and 1,813,000 cell/mL milk, respectively. Milk yield and days in milk did not differ among the 3 groups. Bacterial isolates included coagulase-negative staphylococci, Escherichia coli, Streptococcus dysgalactiae, or no bacteria found. Ovaries were collected at the abattoir and brought to the laboratory. Cumulus oocyte complexes were recovered separately from each cow and subjected individually to in vitro maturation and fertilization, followed by 8d in culture. The number of aspirated oocytes did not differ among groups, with a range of 17 to 21 oocytes per cow. The proportion of oocytes that cleaved into 2- to 4-cell-stage embryos (86.1 ± 3.4%) did not differ among groups. In contrast, mean percentages of embryos developed to the blastocyst stage on d 7 and 8 after fertilization were less in both medium- and-high SCC groups than in the low-SCC group (5.6 ± 2.3 and 4.1 ± 1.8 vs. 18.1 ± 4.6%, respectively). Additional analysis indicated that cleavage and blastocyst-formation rates did not differ among the bacterial types in the low-, medium-, and high-SCC groups. These are the first results to demonstrate that naturally occurring mastitis disrupts the developmental competence of the ovarian pool of oocytes, (i.e., oocytes at the germinal vesicle stage). The disruption was associated with elevation of SCC rather than bacterial type. The results may provide a partial explanation for the low fertility of cows that have contracted mastitic pathogens before insemination. PMID:23957998

  17. 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...

  18. Ground Penetrating Radar, Barrow, Alaska

    SciTech Connect

    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.

  19. 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.

  20. Protein expression profile of the mouse metaphase-II oocyte.

    PubMed

    Ma, Minyue; Guo, Xuejiang; Wang, Fuqiang; Zhao, Chun; Liu, Zichuan; Shi, Zhonghua; Wang, Yufeng; Zhang, Ping; Zhang, Kemei; Wang, Ningling; Lin, Min; Zhou, Zuomin; Liu, Jiayin; Li, Qingzhang; Wang, Liu; Huo, Ran; Sha, Jiahao; Zhou, Qi

    2008-11-01

    The mature oocyte contains the full complement of maternal proteins required for fertilization, the transition to zygotic transcription, and the beginning stages of embryogenesis. Many of these proteins have yet to be characterized. In this study, two-dimensional electrophoresis (2-DE) of mouse metaphase-II (MII) oocyte proteins, stained with silver staining or Pro-Q Diamond dye, was performed to describe the proteome and phosphoproteome of the mouse oocyte derived from ICR mice. A total of 869 selected protein spots, corresponding to 380 unique proteins, were identified successfully by mass spectrometry, in which 90 protein spots representing 53 unique proteins have been stained with Pro-Q Diamond, indicating that they are in phosphorylated forms. All identified proteins were bioinformatically annotated in detail and compared with the embryonic stem cell (ESC) proteome. A proteome reference database for the mouse oocyte was established from the protein data generated in this study, which can be accessed over the Internet ( http://reprod.njmu.edu.cn/2d). This database is the most detailed mouse oocyte proteomic database to date. It should be valuable in expanding our knowledge of the regulation of signaling in oogenesis, fertilization, and embryo development, while revealing potential mechanisms for epigenetic reprogramming. PMID:18803416

  1. 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.

  2. Storage of Steindachneridion parahybae oocytes at different temperatures.

    PubMed

    Sanches, Eduardo Antônio; Okawara, Renan Yoshiharu; Caneppele, Danilo; Neumann, Giovano; Bombardelli, Robie Allan; Romagosa, Elizabeth

    2014-12-30

    The objective of this study was to assess the influence of temperature and time on the storage of fresh Steindachneridion parahybae oocytes. Two experiments were carried out: (1) the fertilization rates of oocytes exposed to temperatures of 5, 15, 28 (room temperature) and 35°C were assessed 15min (control), 115, 235 and 355min after release; (2) the fertilization and hatching rates, as well as the percentage of normal larvae of oocytes exposed to 14, 17 or 20°C, 20min (control) were assessed 50, 80 and 110min after stripping. In the first experiment, the highest fertilization rates (P<0.05) were obtained in the control treatment (15min, 28°C), with 74.34±5.48% oocytes showing loss of viability over time. In the second experiment, there was a reduction (P<0.05) in the fertilization rates at the temperatures and times tested. The artificial fertilization of S. parahybae oocytes is recommended immediately after collection, and if storage is necessary, it should be conducted at temperatures between 17 and 20°C. PMID:25458322

  3. PLK4 is essential for meiotic resumption in mouse oocytes.

    PubMed

    Luo, Yi-Bo; Kim, Nam-Hyung

    2015-04-01

    Polo-like kinase (PLK) 4 is a unique member of the PLK family that plays vital roles in centriole biogenesis during mitosis. The localization of PLK4 on centrioles must be precisely regulated during mitosis to ensure correct centriole duplication. However, little is known about the function of PLK4 in mammalian oocyte meiosis. We addressed this question by examining the expression and localization of PLK4 in mouse oocytes and using RNA interference and protein overexpression to investigate its function in meiosis. PLK4 expression peaked at the germinal vesicle breakdown (GVBD) stage, and the protein was localized in the cytoplasm throughout meiotic maturation. Depletion of PLK4 caused meiotic arrest at the GV stage and suppressed CYCLINB1 and CDC2 activities. Moreover, PLK4 depletion prevented the de-phosphorylation of CDC2-Tyr15 in nucleus and induced a decrease in the level of the CDC25C protein. PLK1 overexpression failed to rescue GV-stage arrest in PLK4-depleted oocytes, whereas overexpressing PLK4 resulted in normal GVBD in oocytes in which PLK1 activity was inhibited. In addition, PLK4 overexpression did not cause abnormal spindle formation or affect extrusion of the first polar body. These results illustrate the fact that PLK4 is essential for meiotic resumption but may not influence spindle formation in mouse oocytes during meiotic maturation. PMID:25740542

  4. 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

  5. Penetrant-Indication-Measuring Compass

    NASA Technical Reports Server (NTRS)

    Schaefer, Lloyd

    1991-01-01

    Modified drafting compass well suited to measurement of length of crack or width of area stained by penetrant-dye-inspection method. Equipped with any of variety of standard curved or straight pointed tips. Modification consists in coating tips with dye that fluoresces light pink under same ultraviolet inspection light causing penetrant dye to fluoresce yellow green. Used in locations inaccessible to conventional fluorescent comparator. Eliminates errors of optical distortion in comparator, also eliminates errors of interpolation.

  6. 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

  7. Demonstration of Survivable Space Penetrator

    NASA Astrophysics Data System (ADS)

    Church, P.; Huntington-Thresher, W.; Penny, N.; Bruce, A.; Smith, A.; Gowan, R.

    2009-06-01

    This work was performed in support of MoonLITE which is a proposed UK space mission to the moon. The basic premise is to deploy 4 instrumented penetrators, one each on the near-side, far-side and at the poles of the moon, with an impact velocity of approximately 300m/s. The primary science aims are to set up a passive seismometer network, investigate the presence of water and volatiles and determine thermal gradients in the lunar soil (i.e. regolith). A key requirement is that the penetrator shell survives the impact together with the instrument payload and supporting subsystems. The material chosen for the penetrator shell was 7075 aluminum alloy, which is a good compromise between high compressive strength and low mass. The baseline penetrator design was evaluated and refined using the DYNA3D hydrocode to determine the survivability of the penetrator in sand at an impact velocity of 300m/s and an attack angle of 8 degrees. The simulations predicted that the penetrator design would survive this severe impact condition which was confirmed by experiments on the Pendine rocket test track.

  8. Demonstration of survivable space penetrator

    NASA Astrophysics Data System (ADS)

    Church, Philip; Huntington-Thresher, William; Bruce, Alan; Penny, Nick; Smith, Alan; Gowan, Rob

    2012-03-01

    This work was performed in support of MoonLITE which is a proposed UK space mission to the moon. The basic premise is to deploy 4 instrumented penetrators, one each on the near-side, farside and at the poles of the moon, with an impact velocity of approximately 300m/s. The primary science aims are to set up a passive seismometer network, investigate the presence of water and volatiles and determine thermal gradients in the lunar soil (i.e. regolith). A key requirement is that the penetrator shell survives the impact together with the instrument payload and supporting subsystems. The material chosen for the penetrator shell was 7075 aluminium alloy, which is a good compromise between high compressive strength and low mass. The baseline penetrator design was evaluated and refined using the DYNA3D hydrocode to determine the survivability of the penetrator in sand at an impact velocity of 300m/s and an attack angle of 8. The simulations predicted that the penetrator design would survive this severe impact condition which was confirmed by experiments on the Pendine rocket test track.

  9. 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 ?m diameter nucleolinus within the oocyte nucleolus. Oocytes were the followed and assayed for polar body ejection.

  10. Modified vitrification of human pronuclear oocytes: efficacy and effect on ultrastructure.

    PubMed

    Isachenko, Vladimir; Selman, Helmy; Isachenko, Eugenia; Montag, Markus; El-Danasouri, Imam; Nawroth, Frank

    2003-09-01

    The efficacy of cryopreservation by direct plunging into liquid nitrogen (vitrification) of human pronuclear oocytes using open pulled straws with a super-finely pulled tip, as well as the ultrastructural changes caused by cooling and vitrification, were evaluated. Clinical and electron microscopic studies of cooled and vitrified oocytes were performed. Oocytes were cooled to 4 degrees C in the presence and absence of cryoprotectants, vitrified, warmed, cultured and transferred. Abnormally fertilized oocytes were examined by electron microscopy. Vitrified and warmed 2-pronuclear oocytes showed 71.1% survival rate and 83.3% developmental rate. One- and 3-pronuclear oocytes, after cooling without cryoprotectants (presumably non-viable), showed progressive swelling of mitochondrial smooth endoplasmic reticulum (SER). After vitrification, oocytes (presumably viable) showed the formation of large SER vesicles associated with mitochondria. The described protocol of vitrification of human pronuclear oocytes was shown to be effective in producing pregnancy. Normal ultrastructure after undergoing the described vitrification protocol was confirmed. PMID:14567894

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

    PubMed Central

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

    2012-01-01

    Abstract. 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 ?m diameter nucleolinus within the oocyte nucleolus. Oocytes were the followed and assayed for polar body ejection. PMID:22352645

  12. Signalling pathways involved in oocyte growth, acquisition of competence and activation.

    PubMed

    Nunes, Cludia; Silva, Joana Vieira; Silva, Vladimiro; Torgal, Isabel; Fardilha, Margarida

    2015-06-01

    The oocyte's primary function is to be fertilised by a spermatozoon in order to create a viable embryo. Oocyte growth and development are initiated during embryogenesis and occur in parallel to follicular development. Factors produced by the oocyte bind to receptors on follicular cells, ensuring follicular development. Oocytes begin meiosis during foetal development and are arrested in prophase I by elevated levels of cyclic adenosine monophosphate (cAMP). Activation of mitogen-activated protein kinases triggers degradation of cAMP, allowing oocyte maturation to proceed. The production of progesterone and prostaglandins during the ovulation process ultimately activates proteases, whose action helps to release the oocyte into the Fallopian tube. Oocyte activation depends on fertilisation and is induced by changes in intracellular calcium levels. Dysregulation of these pathways is involved in the pathogenesis of several diseases including the syndrome of oocyte maturation failure. PMID:25738216

  13. Meiotically incompetent and competent goat oocytes: timing of nuclear events and protein phosphorylation.

    PubMed

    Gall, L; De Smedt, V; Crozet, N; Ruffini, S; Svellec, C

    1996-10-01

    The ability of mammalian oocytes to resume meiosis and to complete the first meiotic division is acquired sequentially during their growth phase. The acquisition of meiotic competence in goat oocytes has been previously correlated with follicular size (9). Since protein phosphorylation/dephosphorylation play a key role in oocyte maturation, it could be that in meiotically incompetent oocytes, such post-translational modifications are inadequate. The aim of this study was to analyze whether changes in oocyte proteins phosphorylation occurred during the acquisition of meiotic competence. For this propose, goat oocytes were divided into 4 classes according to follicular size and meiotic competence: Class A oocytes from follicles < 0.5 mm in diameter: Class B oocytes from follicles 0.5-0.8 mm; Class C oocytes from follicles 1-1.8 mm and class D oocytes from follicles > 3 mm. The protein phosphorylation patterns of these classes of oocytes were studied at different times of in vitro maturation. After 4h of culture, when all oocytes were in the germinal vesicle stage, only the oocytes from Class D displayed the phosphoproteins at 110 kD, 31 kD and around 63 kD. In contrast to Class D oocytes Classes B and C oocytes were partially competent to mature, they underwent germinal vesicle breakdown later than fully competent Class D oocytes and remained in early prometaphase I or in metaphase I, respectively. They exhibited the phosphoprotein changes that are associated with commitment to resume meiosis; but the changes occurred later than in Class D oocytes, which were fully competent to reach metaphase II. After 27 h of culture, the phosphorylation patterns of Class B, C and D oocytes were identical, whereas the meiotic stages reached were quite different. The phosphoprotein changes associated with oocyte maturation did not occur in meiotically incompetent Class A oocytes, which were blocked at the germinal vesicle stage. From these results it can be concluded that, at the GV stage, meiotically incompetent and competent goat oocytes display different patterns of protein phosphorylation. Once oocytes are able to resume meiosis they undergo specific phosphorylation changes, but whether these changes are markers or regulators of maturation events remains to be determined. PMID:16727946

  14. 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.

  15. Hamsters' (Mesocricetus auratus) memory in a radial maze analog: the role of spatial versus olfactory cues.

    PubMed

    Tonneau, François; Cabrera, Felipe; Corujo, Alejandro

    2012-02-01

    The golden hamster's (Mesocricetus auratus) performance on radial maze tasks has not been studied a lot. Here we report the results of a spatial memory task that involved eight food stations equidistant from the center of a circular platform. Each of six male hamsters depleted the food stations along successive choices. After each choice and a 5-s retention delay, the hamster was brought back to the center of the platform for the next choice opportunity. When only one baited station was left, the platform was rotated to evaluate whether olfactory traces guided hamsters' choices. Results showed that despite the retention delay hamsters performed above chance in searching for food. The choice distributions observed during the rotation probes were consistent with spatial memory and could be explained without assuming guidance by olfactory cues. The radial maze analog we devised could be useful in furthering the study of spatial memory in hamsters. PMID:21842978

  16. Autoradiography in fetal golden hamsters treated with tritiated diethylnitrosamine

    SciTech Connect

    Reznik-Schueller, H.M.; Hague, B.F. Jr.

    1981-04-01

    Tritiated diethylnitrosamine was administered to female Syrian golden hamsters on each of the last 4 days (days 12-15) of pregnancy. The distribution of bound radioactivity was monitored by light microscopic autoradiography of fetal tracheas and livers, the placentas, and the maternal livers. In the trachea, the fetal target organ, bound radioactivity was restricted to the respiratory epithelium, where diethylnitrosamine-induced tracheal tumors arise. Mucous cells and nonciliated stem cells were identified as the principal sites of binding; other cell types within the tracheal epithelium contained only small amounts of bound radioactivity. The level of binding observed in the fetal trachea increased steadily from day 12 to day 15, which correlated well with the levels of differentiation of this tissue during this period. This observation also agrees with the previously reported observation that tumor incidence increases from 40 to 95% in Syrian golden hamsters between days 12 and 15.

  17. Identification and characterization of the hamster polyomavirus middle T antigen.

    PubMed Central

    Courtneidge, S A; Goutebroze, L; Cartwright, A; Heber, A; Scherneck, S; Feunteun, J

    1991-01-01

    Hamster polyomavirus (HaPV) is associated with lymphoid and hair follicle tumors in Syrian hamsters. The early region of HaPV has the potential to encode three polypeptides (which are related to the mouse polyomavirus early proteins) and can transform fibroblasts in vitro. We identified the HaPV middle T antigen (HamT) as a 45-kDa protein. Like its murine counterpart, HamT was associated with serine/threonine phosphatase, phosphatidylinositol-3 kinase, and protein tyrosine kinase activities. However, whereas mouse middle T antigen associates predominantly with pp60c-src and pp62c-yes, HamT was associated with a different tyrosine kinase, p59fyn. The ability of HaPV to cause lymphoid tumors may therefore reside in its ability to associate with p59fyn, a potentially important tyrosine kinase in lymphocytes. Images PMID:1709702

  18. Oxidative metabolites of diethylstilbestrol in the fetal Syrian golden hamster

    SciTech Connect

    Maydl, R.; Metzler, M.

    1984-12-01

    /sup 14/C-Diethylstilbestrol was administered orally, intraperitoneally, and intrafetally to 15-day pregnant hamsters at a dose of 20 mg/kg body weight, and the radioactivity was determined in the fetus, placenta, and maternal liver after 6 hours. Significant amounts of radioactivity were found in these tissues in every case, indicating maternal-fetal and fetal-maternal transfer of diethylstilbestrol. Part of the radioactivity found in the tissues could not be extracted even after excessive washing. This implied the presence of reactive metabolites. In the fetal and placental extracts, eight oxidative metabolites of diethylstilbestrol were identified by mass fragmentography as hydroxy- and methoxy-derivatives of diethylstilbestrol, pseudodiethylstilbestrol, and dienestrol. The presence of oxidative metabolites in the hamster fetus and the covalent binding to tissue macromolecules are possibly associated with the fetotoxic effects of diethylstilbestrol.

  19. 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.

  20. Highly Virulent Leptospira borgpetersenii Strain Characterized in the Hamster Model

    PubMed Central

    Diniz, Juliana Alcoforado; Flix, Samuel Rodrigues; Bonel-Raposo, Josiane; Seixas Neto, Amilton Clair Pinto; Vasconcellos, Flvia Aleixo; Grassmann, Andr Alex; Dellagostin, Odir Antnio; Aleixo, Jos Antonio Guimares; da Silva, verton Fagonde

    2011-01-01

    A recent study by our group reported the isolation and partial serological and molecular characterization of four Leptospira borgpetersenii serogroup Ballum strains. Here, we reproduced experimental leptospirosis in golden Syrian hamsters (Mesocricetus auratus) and carried out standardization of lethal dose 50% (LD50) of one of these strains (4E). Clinical disease features and histopathologic analyses of tissue lesions were also observed. As results, strain 4E induced lethality in the hamster model with inocula lower than 10 leptospires, and histopathological examination of animals showed typical lesions found in severe leptospirosis. Gross pathological findings were peculiar; animals that died early had more chance of presenting severe jaundice and less chance of presenting pulmonary hemorrhages (P < 0.01). L. borgpetersenii serogroup Ballum has had a considerable growth in human leptospirosis cases in recent years. This strain has now been thoroughly characterized and can be used in more studies, especially evaluations of vaccine candidates. PMID:21813846

  1. Mitochondrial Distribution and ATP Content of Vitrified, In vitro Matured Mouse Oocytes

    PubMed Central

    Nazmara, Zohreh; Salehnia, Mojdeh; HosseinKhani, Saman

    2014-01-01

    Background The objective of this study was to investigate the effect of vitrification and in vitro maturation on the mitochondrial distribution and ATP content of oocytes. Methods The oocytes at Germinal Vesicle (GV) and Metaphase II (MII) stages were recovered from 6-8 week old NMRI strain female mice. The oocytes were divided into vitrified and non-vitrified groups. Vitrification was done by the cryotop method using ethylene glycol, dimethylsulfoxide and sucrose as cryoprotectants. The GV oocytes were cultured in maturation medium for 24 hrs. The collected in vitro matured oocytes (IVM-MII) and ovulated metaphase II (OV-MII) oocytes were inseminated with capacitated sperm. The ATP content of the oocytes was measured by luciferin-luciferase reaction. Distribution of oocyte mitochondria was studied using Mito Tracker Green staining under fluorescent microscope. Results The survival rates of vitrified oocytes at GV and MII stages were 87.39 and 89.5%, respectively. There was no significant difference in the developmental and hatching rates of vitrified and non-vitrified oocytes. The ATP content of GV and MII oocytes derived from in vivo and in vitro condition was not significantly different in vitrified and non-vitrified samples. The pattern of mitochondrial distribution in vitrified and non-vitrified GV and MII oocytes was similar but it was different between MII oocytes collected from fallopian tube and in vitro matured MII oocytes. However, the florescent intensity of mitochondrial staining was different in all the groups in the study. Conclusion Vitrification did not affect mouse oocyte developmental competence, ATP content at different developmental stages but some alteration was seen in mitochondria distribution of in vitro matured oocytes in comparison to their controls. PMID:25414783

  2. 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. PMID:23884646

  3. Cumulus and granulosa cell markers of oocyte and embryo quality

    PubMed Central

    Uyar, Asli; Torrealday, Saioa; Seli, Emre

    2013-01-01

    Lack of an objective, accurate, and noninvasive embryo assessment strategy remains one of the major challenges encountered in in vitro fertilization. Cumulus and mural granulosa cells reflect the characteristics of the oocyte, providing a noninvasive means to assess oocyte quality. Specifically, transcriptomic profiling of follicular cells may help identify biomarkers of oocyte and embryo competence. Current transcriptomics technologies include quantitative reverse transcriptase–polymerase chain reaction (qRT-PCR) for analysis of individual genes and microarrays and high-throughput deep sequencing for whole genome expression profiling. Recently, using qRT-PCR and microarray technologies, a multitude of studies correlated changes in cumulus or granulosa cell gene expression with clinically relevant outcome parameters, including in vitro embryo development and pregnancy. While the initial findings are promising, a clinical benefit from the use of identified biomarker genes remains to be demonstrated in randomized controlled trials. PMID:23498999

  4. 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

  5. Live imaging of GFP-labeled proteins in Drosophila oocytes.

    PubMed

    Pokrywka, Nancy Jo

    2013-01-01

    The Drosophila oocyte has been established as a versatile system for investigating fundamental questions such as cytoskeletal function, cell organization, and organelle structure and function. The availability of various GFP-tagged proteins means that many cellular processes can be monitored in living cells over the course of minutes or hours, and using this technique, processes such as RNP transport, epithelial morphogenesis, and tissue remodeling have been described in great detail in Drosophila oocytes. The ability to perform video imaging combined with a rich repertoire of mutants allows an enormous variety of genes and processes to be examined in incredible detail. One such example is the process of ooplasmic streaming, which initiates at mid-oogenesis. This vigorous movement of cytoplasmic vesicles is microtubule and kinesin-dependent and provides a useful system for investigating cytoskeleton function at these stages. Here I present a protocol for time lapse imaging of living oocytes using virtually any confocal microscopy setup. PMID:23567977

  6. Expanding the genetic code in Xenopus laevis oocytes.

    PubMed

    Ye, Shixin; Riou, Morgane; Carvalho, Stphanie; 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

  7. Oocyte quality of tambaqui (Colossoma macropomum) during the reproductive season.

    PubMed

    Galo, J M; Ribeiro, R P; Streit-Junior, D P; Albuquerque, D M; Fornari, D C; Roma, C F C; Guerreiro, L R J

    2015-05-01

    The study aimed to analyze the Colossoma macropomum reproductive behavior and quality of the female gametes throughout the reproductive season. The experiment was carried out in Pimenta Bueno - Rondônia State (Northern Brazil) during the reproductive season (2010-2011) using 36 females. Each sampling was performed on a 15 ± 5 days interval. Female gametes were collected by stripping and the following analyses were performed: weight of oocytes released (g); productivity index, fertilization and hatching rate. During the sampling period was verified effect (p < 0.05) of collecting time into the season for oocytes weight, productivity index and fertilization rate. Although the period 3 (December) did not differ significantly from other periods, it showed better parameters for the quality of C. macropomum oocytes. PMID:26132008

  8. Cryopreservation of embryos and oocytes in human assisted reproduction.

    PubMed

    Konc, Jnos; Kany, Katalin; Kriston, Rita; Somosk?i, Bence; Cseh, Sndor

    2014-01-01

    Both sperm and embryo cryopreservation have become routine procedures in human assisted reproduction and oocyte cryopreservation is being introduced into clinical practice and is getting more and more widely used. Embryo cryopreservation has decreased the number of fresh embryo transfers and maximized the effectiveness of the IVF cycle. The data shows that women who had transfers of fresh and frozen embryos obtained 8% additional births by using their cryopreserved embryos. Oocyte cryopreservation offers more advantages compared to embryo freezing, such as fertility preservation in women at risk of losing fertility due to oncological treatment or chronic disease, egg donation, and postponing childbirth, and eliminates religious and/or other ethical, legal, and moral concerns of embryo freezing. In this review, the basic principles, methodology, and practical experiences as well as safety and other aspects concerning slow cooling and ultrarapid cooling (vitrification) of human embryos and oocytes are summarized. PMID:24779007

  9. 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

  10. Central vs. peripheral metabolic control of estrous cycles in Syrian hamsters. I. Lipoprivation.

    PubMed

    Schneider, J E; Hall, A J; Wade, G N

    1997-01-01

    Metabolic energy availability has profound effects on reproduction in a wide variety of species. We have been studying the effects of fasting on estrous cycles in Syrian hamsters as a model system for metabolic control of reproduction. In previous experiments, a 48-h period of fasting inhibited estrous cycles in lean, but not fat, hamsters. In fat hamsters the effects of fasting may have been offset by the presence of high circulating levels of free fatty acids mobilized from lipids in adipose tissue. Consistent with this idea fat hamsters treated with the inhibitor of fatty acid oxidation methyl palmoxirate (MP) showed fasting-induced anestrus. Experiment 1 was designed to examine whether vagally transmitted signals are critical for the inhibitory effects of fasting and MP treatment. Lean or fat hamsters that had received bilateral subdiaphragmatic vagotomy or sham surgery were fasted and treated with MP or vehicle. In vagotomized and sham-operated hamsters, estrous cycles were inhibited in lean fasted hamsters and in fat fasted hamsters treated with MP, but not in fat fasted hamsters treated with vehicle. Thus the results of experiment 1 indicated that vagally transmitted signals about peripheral fatty acid availability are not critical for the effects of these particular metabolic challenges on estrous cycles in Syrian hamsters. In experiment 2, hamsters without food were allowed to ingest pure glucose or fructose solutions or vegetable shortening. One-half of each group was treated with an inhibitor of glucose utilization, 2-deoxy-D-glucose (2-DG), or vehicle. If ingestion of fructose or shortening, but not glucose, had protected hamsters from 2-DG-induced anestrus, this might have indicated that peripheral fuel availability is critical for anestrus. On the contrary, 2-DG treatment induced anestrus regardless of the type of fuel ingested. Neither experiment yielded results that implicated changes in peripheral fuel availability as a critical signal in metabolic control of estrous cycles. PMID:9039035

  11. 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.

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

    PubMed Central

    Torres, Jos Alberto; de las Heras, Manuel; Maroto, Aroa Sanz; Vivanco, Fernando; Sastre, Joaqun; 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

  13. Selective decontamination of the digestive tract of Syrian hamsters.

    PubMed

    Angulo, A F; Spaans, J; Zemmouchi, L; van der Waaij, D

    1978-07-01

    Conventional Syrian hamsters colonized with aerobic gram-negative bacteria such as Pasteurella pneumotropica and various Enterobacteriaceae species were successfully and permanently freed from these microorganisms by oral treatment for 4 weeks with dihydrostreptomycin and 'Orabase' premixed with appropriate antibiotics. Concomitant oral treatment with dimetridazol for the elimination of intestinal flagellates was unsuccessful. During treatment the animals were maintained under germ-free isolation conditions. PMID:82636

  14. 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-1? (IL-1?) and indoleamine 2,3-dioxygenase (Ido) mRNA expression in aged hamsters, but social housing decreased hippocampal IL-1? 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-1? and Ido activity, which may contribute to the ameliorating effects of social housing on a number of emotional behaviors. PMID:24333374

  15. Expression of TGF? superfamily components and other markers of oocyte quality in oocytes selected by brilliant cresyl blue staining: Relevance to early embryonic development

    PubMed Central

    Ashry, Mohamed; Lee, KyungBon; Mondal, Mohan; Datta, Tirtha K.; Folger, Joseph K.; Rajput, Sandeep K.; Zhang, Kun; Hemeida, Nabil A.; Smith, George W.

    2015-01-01

    Brilliant cresyl blue (BCB) is a super vital stain that has been used to select competent oocytes in different species. The objectives of present studies were to determine mRNA abundance for select TGF? superfamily components, SMAD2/3 and SMAD1/5 phosphorylation levels and transcript abundance for other oocyte (JY1) and cumulus cell (CTSB, CTSK, CTSS and CTSZ) markers of oocyte quality in bovine oocytes and or adjacent cumulus cells classified based on developmental potential using BCB staining. The ability of exogenous FST, JY1, or cathepsin inhibitor treatment to enhance development of embryos derived from poor quality oocytes selected based on BCB staining was also determined. Cumulus oocyte complexes (COCs) from abattoir derived ovaries were subjected to BCB staining and GV stage oocytes and cumulus cells harvested from control, BCB+ and BCB- (poor oocyte quality) groups for real time PCR or Western blot analysis. Remaining COCs underwent in vitro maturation, in vitro fertilization and embryo culture in presence or absence of above described treatments. Levels of FST, JY1, BMP15 and SMAD1, 2, 3 and 5 transcripts were higher in BCB+ oocytes whereas abundance of CTSB, CTSK, CTSS and CTSZ mRNAs was higher in cumulus cells surrounding poor quality BCB- oocytes. Western blot analysis revealed SMAD1/5 and SMAD2/3 phosphorylation were higher in BCB+ than BCB? oocytes. Embryo culture studies demonstrated that follistatin and cathepsin inhibitor treatment but not JY-1 treatment can promote developmental competence of BCB- oocytes. Results provide further understanding of molecular indices of oocyte competence. PMID:25704641

  16. Expression of TGF? superfamily components and other markers of oocyte quality in oocytes selected by brilliant cresyl blue staining: relevance to early embryonic development.

    PubMed

    Ashry, Mohamed; Lee, KyungBon; Mondal, Mohan; Datta, Tirtha K; Folger, Joseph K; Rajput, Sandeep K; Zhang, Kun; Hemeida, Nabil A; Smith, George W

    2015-03-01

    Brilliant cresyl blue (BCB) is a super-vital stain that has been used to select competent oocytes in different species. One objective of the present study was to assess the relationship between BCB staining, which correlates with an oocyte's developmental potential, and the transcript abundance for select TGF?-superfamily components, SMAD2/3 and SMAD1/5 phosphorylation levels, and oocyte (JY1) and cumulus-cell (CTSB, CTSK, CTSS, and CTSZ) transcript markers in bovine oocytes and/or adjacent cumulus cells. The capacity of exogenous follistatin or JY1 supplementation or cathepsin inhibitor treatment to enhance development of embryos derived from low-quality oocytes, based on BCB staining, was also determined. Cumulus-oocyte complexes (COCs) from abattoir-derived ovaries were subjected to BCB staining, and germinal-vesicle-stage oocytes and cumulus cells were harvested from control, BCB+, and BCB- (low-quality oocyte) groups for real-time PCR or Western-blot analysis. Remaining COCs underwent in vitro maturation, in vitro fertilization, and embryo culture in the presence or absence of the above exogenous supplements. Levels of FST, JY1, BMP15, and SMAD1, 2, 3, and 5 transcripts were higher in BCB+ oocytes whereas CTSB, CTSK, CTSS, and CTSZ mRNA abundance was higher in cumulus cells surrounding BCB- oocytes. Western-blot analysis revealed higher SMAD1/5 and SMAD2/3 phosphorylation in BCB+ than BCB- oocytes. Embryo-culture studies demonstrated that follistatin and cathepsin inhibitor treatment, but not JY-1 treatment, improve the developmental competence of BCB- oocytes. These results contribute to a better understanding of molecular indices of oocyte competence. PMID:25704641

  17. 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

  18. 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

  19. Cholesterol Depletion Disorganizes Oocyte Membrane Rafts Altering Mouse Fertilization

    PubMed Central

    Buschiazzo, Jorgelina; Ialy-Radio, Come; Auer, Jana; Wolf, Jean-Philippe; Serres, Catherine

    2013-01-01

    Drastic membrane reorganization occurs when mammalian sperm binds to and fuses with the oocyte membrane. Two oocyte protein families are essential for fertilization, tetraspanins and glycosylphosphatidylinositol-anchored proteins. The firsts are associated to tetraspanin-enriched microdomains and the seconds to lipid rafts. Here we report membrane raft involvement in mouse fertilization assessed by cholesterol modulation using methyl-?-cyclodextrin. Cholesterol removal induced: (1) a decrease of the fertilization rate and index; and (2) a delay in the extrusion of the second polar body. Cholesterol repletion recovered the fertilization ability of cholesterol-depleted oocytes, indicating reversibility of these effects. In vivo time-lapse analyses using fluorescent cholesterol permitted to identify the time-point at which the probe is mainly located at the plasma membrane enabling the estimation of the extent of the cholesterol depletion. We confirmed that the mouse oocyte is rich in rafts according to the presence of the raft marker lipid, ganglioside GM1 on the membrane of living oocytes and we identified the coexistence of two types of microdomains, planar rafts and caveolae-like structures, by terms of two differential rafts markers, flotillin-2 and caveolin-1, respectively. Moreover, this is the first report that shows characteristic caveolae-like invaginations in the mouse oocyte identified by electron microscopy. Raft disruption by cholesterol depletion disturbed the subcellular localization of the signal molecule c-Src and the inhibition of Src kinase proteins prevented second polar body extrusion, consistent with a role of Src-related kinases in fertilization via signaling complexes. Our data highlight the functional importance of intact membrane rafts for mouse fertilization and its dependence on cholesterol. PMID:23638166

  20. Ultrastructural markers of quality in human mature oocytes vitrified using cryoleaf and cryoloop.

    PubMed

    Nottola, S A; Coticchio, G; Sciajno, R; Gambardella, A; Maione, M; Scaravelli, G; Bianchi, S; Macchiarelli, G; Borini, A

    2009-01-01

    This study describes and compares the possible effects of vitrification on the ultrastructural morphology of 20 human mature oocytes vitrified using two different supports, cryoleaf (n = 10) and cryoloop (n = 10). Fresh human mature oocytes (n = 15) were used as controls. Fresh and vitrified-warmed oocytes appeared rounded, with a homogeneous cytoplasm, an intact oolemma and a continuous zona pellucida. Sparse microvacuolization was only occasionally detected in fresh and vitrified-warmed oocytes, to the same extent. About 50% of the vitrified oocytes contained atypical, small and slender mitochondria-smooth endoplasmic reticulum aggregates, whereas a non-homogeneous microvillar pattern was observable in only 30% of the oocytes subjected to vitrification, regardless of the support utilized. Cortical granule content appeared generally reduced after vitrification, but cryoleaf-supported oocytes contained more cortical granules than cryoloop-supported oocytes (P < 0.05). Thus good overall preservation and virtual absence of cytoplasmic vacuolization seem to be the most relevant markers of quality in vitrified-warmed oocytes, using either support. In addition, cryoleaf-supported oocytes retained a higher number of cortical granules than cryoloop-supported oocytes. The variety of ultrastructural alterations recorded emphasizes the need for further studies aimed at assessing the actual tolerance of human oocytes to vitrification. PMID:20034420

  1. 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

  2. Assessment of meiotic spindle configuration and post-warming bovine oocyte viability using polarized light microscopy.

    PubMed

    Caamao, J N; Dez, C; Trigal, B; Muoz, M; Morat, R; Martn, D; Carrocera, S; Mogas, T; Gmez, E

    2013-06-01

    The objectives of this study were to assess the efficiency of polarized light microscopy (PLM) in detecting microtubule-polymerized protein in in vitro-matured bovine oocytes; to examine its effects on oocyte developmental competence; and to assess the meiotic spindle of in vitro-matured oocytes after vitrification/warming and further assessment of oocyte developmental competence. In the first experiment, the presence of microtubule-polymerized protein (MPP) was confirmed as a positive PLM signal detected in 99.1% of analysed oocytes (n=115), which strongly correlated (r=1; p<0.0001) with the presence of MPP as confirmed by immunostaining. In the second experiment, oocytes (n=651) were exposed or not (controls) to PLM for 10min and then fertilized and cultured in vitro. Oocytes exposed to PLM did not significantly differ from controls with regard to cleavage, total blastocyst and expanded blastocyst rates and cell numbers. In the third experiment, meiotic spindles were detected in 145 of 182 oocytes (79.6%) following vitrification and warming. Interestingly, after parthenogenetic activation and in vitro culture, oocytes that displayed a positive PLM signal PLM(+) differed significantly from PLM(-) in cleavage and Day 8 blastocyst rates. These results suggest that polarized light microscopy is an efficient system to detect microtubule-polymerized protein in in vitro-matured bovine oocytes and does not exert detrimental effects on bovine oocyte developmental competence. Moreover, PLM could be used as a tool to assess post-warming viability in vitrified bovine oocytes. PMID:23106568

  3. Use of oocytes from anonymous, matched, fertile donors for prevention of heritable genetic diseases.

    PubMed Central

    Van Voorhis, B J; Williamson, R A; Gerard, J L; Hammitt, D G; Syrop, C H

    1992-01-01

    Heritable genetic diseases can be prevented with the use of donor oocytes. We report our experience in using donor oocytes from anonymous, matched, fertile donors in four women with heritable genetic disorders. Our results show that use of donor oocytes is a practical, successful, and currently available technique for the prevention of genetic disorders. PMID:1619634

  4. 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. PMID:24866911

  5. 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.

  6. Isolation of two chloroethylnitrosourea-sensitive Chinese hamster cell lines

    SciTech Connect

    Hata, H.; Numata, M.; Tohda, H.; Yasui, A.; Oikawa, A. )

    1991-01-01

    1-((4-Amino-2-methylpyrimidin-5-yl)methyl)-3-(2-chloroethyl)-3- nitrosourea hydrochloride (ACNU), a cancer chemotherapeutic bifunctional alkylating agent, causes chloroethylation of DNA and subsequent DNA strand cross-linking through an ethylene bridge. We isolated and characterized two ACNU-sensitive mutants from mutagenized Chinese hamster ovary cells and found them to be new drug-sensitive recessive Chinese hamster mutants. Both mutants were sensitive to various monofunctional alkylating agents in a way similar to that of the parental cell lines CHO9. One mutant (UVS1) was cross-sensitive to UV and complemented the UV sensitivity of all Chinese hamster cell lines of 7 established complementation groups. Since UV-induced unscheduled DNA synthesis was very low, a new locus related to excision repair is thought to be defective in this cell line. Another ACNU-sensitive mutant, CNU1, was slightly more sensitive to UV than the parent cell line. CNU1 was cross-sensitive to 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea and slightly more sensitive to mitomycin C. No increased accumulation of ACNU and a low level of UV-induced unscheduled DNA synthesis in this cell as compared with the parental cell line suggest that there is abnormality in a repair response of this mutant cell to some types of DNA cross-links.

  7. Histogenesis of pancreatic carcinogenesis in the hamster: ultrastructural evidence

    SciTech Connect

    Flaks, B.

    1984-06-01

    Pancreatic carcinogenesis in the Syrian hamster, induced by ..beta..-oxidized derivatives of N-nitroso-di-n-propylamine, constitutes a valuable model of human cancer of the exocrine pancreas. In both species the majority of tumors are adenocarcinomas: superficially, on the basis of their histological appearance, these appear to be ductal in origin. However, sequential analysis, by electron microscopy, of the development of pancreatic neoplasia in the hamster model indicates that acinar cells may participate in the histogenesis of ductal adenomas and carcinomas. Acinar cells appear to undergo changes in differentiation, including pseudoductular transformation, giving rise to a new population of cells that resemble ductular or centroacinar types. This new population may then proliferate to form, first, cystic foci and subsequently cytadenomas and adenocarcinomas. Mucous metaplasia appears to develop at late stages of tumor development. Although the participation of ductular and centroacinar cells in pancreatic carcinogenesis cannot be excluded, very few tumors arise from the ductal epithelium. It is possible that some human pancreatic adenocarcinomas may also have their origin from dysplastic acinar cells, by analogy with the hamster model: focal acinar dyplasia being common in human pancreatic cancer patients. 90 references, 18 figures.

  8. CHARACTERIZATION OF VIRULENCE OF Leptospira ISOLATES IN A HAMSTER MODEL

    PubMed Central

    Silva, verton F.; Santos, Cleiton S.; Athanazio, Daniel A.; Seyffert, Nbia; Seixas, Fabiana K.; Cerqueira, Gustavo M.; Fagundes, Michel Q.; Brod, Claudiomar S.; Reis, Mitermayer G.; Dellagostin, Odir A.; Ko, Albert I.

    2008-01-01

    Effort has been made to identify protective antigens in order to develop a recombinant vaccine against leptospirosis. Several attempts failed to conclusively demonstrate efficacy of vaccine candidates due to the lack of an appropriate model of lethal leptospirosis. The purposes of our study were: (i) to test the virulence of leptospiral isolates from Brazil, which are representative of important serogroups that cause disease in humans and animals; and (ii) to standardize the lethal dose 50% (LD50) for each of the virulent strains using a hamster (Mesocricetus auratus) model. Five of seven Brazilian isolates induced lethality in a hamster model, with inocula lower than 200 leptospires. Histopathological examination of infected animals showed typical lesions found in both natural and experimental leptospirosis. Results described here demonstrated the potential use of Brazilian isolates as highly virulent strains in challenge experiments using hamster as an appropriate animal model for leptospirosis. Furthermore these strains may be useful in heterologous challenge studies which aim to evaluate cross-protective responses induced by subunit vaccine candidates. PMID:18547690

  9. 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.

  10. 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.

  11. 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, 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 is 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 showed an interesting classification performance. This technique may be of particular interest in those countries where legislation restricts embryo selection. PMID:23177416

  12. Ultrastructural aspects of oocyte maturation and fertilization in cattle.

    PubMed

    Hyttel, P; Greve, T; Callesen, H

    1989-01-01

    The preovulatory surge of LH triggers follicular and oocyte maturation in cattle. Oocyte maturation includes disruption of the gap junctions between cumulus-cell projections and oocyte and the breakdown of the envelope of the oocyte nucleus within 12 h after the LH peak; at approximately 15 h metaphase of the first meiotic division occurs and spatial rearrangements of mitochondria and vesicles are seen in the ooplasm; at approximately 19 h the first polar body is abstricted and the second metaphase appears; and at 21-22 h the cortical granules migrate to solitary positions along the oolemma, the Golgi compartment decreases, and the smooth endoplasmic reticulum (SER) transforms. Ovulations occur in unstimulated and superovulated cattle at approximately 24 h and 24-33 h, respectively. The acrosome reaction, which is preceded by swelling of and appearance of small vesicles in the acrosome, is completed on the surface of the zona pellucida. During the subsequent gamete fusion the microvilli of the ovum contact the equatorial segment of the sperm head, and the acrosomal region is subsequently internalized into the ooplasm surrounded by a vesicle. Within the following 2-3 h the formation of the maternal and paternal pronucleus is initiated, the cortical granules are released, conspicuous Golgi complexes develop, and the SER is transformed; at 5-7 h the pronuclei enlarge, and arrays of annulate lamellae develop. Subsequently, the pronuclei migrate close together; at approximately 20 h the envelopes of the pronuclei are broken down and synkaryosis is seen; and at approximately 24 h the 2-cell stage emerges. Artificial control of oocyte maturation and fertilization in cattle may lead to deviation in these processes. Superovulation may affect oocyte maturation adversely, and in-vitro fertilization may lead to increased frequencies of polyspermy due to deviations in cortical granule release and dispersal. Knowledge about these basal processes of oocyte maturation and fertilization are fundamental in the context of egg manipulation (oocyte enucleation at cloning, gene injection into pronuclei at specific stages etc.) in cattle. PMID:2677348

  13. 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

  14. Acentrosomal Spindle Assembly & Chromosome Segregation During Oocyte Meiosis

    PubMed Central

    Dumont, Julien; Desai, Arshad

    2012-01-01

    The ability to reproduce relies in most eukaryotes on specialized cells called gametes. Gametes are formed by the process of meiosis in which, after a single round of replication, two successive cell divisions reduce the ploidy of the genome. Fusion of gametes at fertilization reconstitutes diploidy. In most animal species, chromosome segregation during female meiosis occurs on spindles assembled in the absence of the major microtubule-organizing center, the centrosome. In mammals, oocyte meiosis is error-prone and underlies the majority of birth aneuploidies. Here, we review recent work on acentrosomal spindle formation and chromosome alignment/separation during oocyte meiosis in different animal models. PMID:22480579

  15. Effect of photoperiod on vasopressin-induced aggression in Syrian hamsters.

    PubMed

    Caldwell, Heather K; Albers, H Elliott

    2004-11-01

    Syrian hamsters are photoperiodic and become sexually quiescent when exposed to short "winter-like" photoperiods. In short photoperiods, male hamsters display significantly higher levels of aggression than males housed in long photoperiods. Arginine-vasopressin (AVP) within the anterior hypothalamus (AH) has been reported to modulate aggression in hamsters housed in long photoperiods. Previous studies have shown that AVP can facilitate aggression and its effects appear to be mediated by AVP V(1a) receptors (V(1a)R). In the present study, we investigated whether the increased levels of aggression observed after exposure to short photoperiod were the result of an increased responsiveness to AVP within the AH. Injections of AVP into the AH significantly increased aggression in hamsters housed in a long photoperiod, but had no effect in hamsters housed in a short photoperiod. In addition, injection of a V(1a)R antagonist into the AH significantly inhibited aggression in hamsters housed in long photoperiod, but had no effect in hamsters housed in a short photoperiod. These findings indicate that AVP within the AH increases aggression in hamsters housed in long photoperiods, but not in hamsters housed in short photoperiods. PMID:15465530

  16. Localization of the Chinese hamster CAD gene reveals homology between human chromosome 2p and Chinese hamster 7q

    SciTech Connect

    Bertoni, L.; Attolini, C.; Giulotto, E. ); Simi, S. )

    1993-06-01

    The trifunctional enzyme CAD catalyzes the first three steps of pyrimidine biosynthesis. By using fluorescence in situ hybridization the authors have localized the Chinese hamster CAD gene on chromosome 7q11-q13 of diploid fibroblasts. Other genes previously assigned to chromosome 7 include acid phosphatase-1, the M2 subunit of ribonucleotide reductase and ornithine decarboxylase. These genes are also syntenic with CAD on human chromosome 2p. They have then mapped CAD on the pericentromeric region of two different rearranged chromosomes (Z8p and R2q) in a cell line derived from Chinese hamster ovary. The presence of CAD on Z8 and R2 indicates that they derive from rearrangements involving chromosome 7. 14 refs., 2 figs.

  17. 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.

  18. 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.

  19. Renal atrial natriuretic factor receptors in hamster cardiomyopathy.

    PubMed

    Mukaddam-Daher, S; Jankowski, M; Dam, T V; Quillen, E W; Gutkowska, J

    1995-12-01

    Hamsters with cardiomyopathy (CMO), an experimental model of congestive heart failure, display stimulated renin-angiotensin-aldosterone and enhanced sympathetic nervous activity, all factors that lead to sodium retention, volume expansion and subsequent elevation of plasma atrial natriuretic factor (ANF) by the cardiac atria. However, sodium and water retention persist in CMO, indicating hyporesponsiveness to endogenous ANF. These studies were undertaken to fully characterize renal ANF receptor subtypes in normal hamsters and to evaluate whether alterations in renal ANF receptors may contribute to renal resistance to ANF in cardiomyopathy. Transcripts of the guanylyl cyclase-A (GC-A) and guanylyl cyclase B (GC-B) receptors were detected by quantitative polymerase chain reaction (PCR) in renal cortex, and outer and inner medullas. Compared to normal controls, the cardiomyopathic hamster's GC-A mRNA was similar in cortex but significantly increased in outer and inner medulla. Levels of GC-B mRNA were not altered by the disease. On the other hand, competitive binding studies, autoradiography, and affinity cross-linking demonstrated the absence of functional GC-B receptors in the kidney glomeruli and inner medulla. Also, C-type natriuretic peptide (CNP), the natural ligand for the GC-B receptors, failed to stimulate glomerular production of its second messenger cGMP. In CMO, sodium and water excretion were significantly reduced despite elevated plasma ANF (50.5 +/- 11.1 vs. 309.4 +/- 32.6 pg/ml, P < 0.001). Competitive binding studies of renal glomerular ANF receptors revealed no change in total receptor density, Bmax (369.6 +/- 27.4 vs. 282.8 +/- 26.2 fmol/mg protein), nor in dissociation constant, Kd (647.4 +/- 79.4 vs. 648.5 +/- 22.9 pM). Also, ANF-C receptor density (254.3 +/- 24.8 vs. 233.8 +/- 23.5 fmol/mg protein), nor affinity were affected by heart failure. Inner medullary receptors were exclusively of the GC-A subtype with Bmax (153.2 +/- 26.4 vs. 134.5 +/- 21.2 fmol/mg protein) and Kd (395.7 +/- 148.0 vs. 285.8 +/- 45.0 pM) not altered by cardiomyopathy. The increase in ANF-stimulated glomerular cGMP production was similar in normal and CMO hamsters (94- vs. 75-fold). These results demonstrate that renal ANF receptors do not contribute to the attenuated renal responses to ANF in hamster cardiomyopathy. PMID:8587247

  20. 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. PMID:21111469

  1. A Western Blot Protocol for Detection of Proteins Heterologously Expressed in Xenopus laevis Oocytes.

    PubMed

    Jrgensen, Morten Egevang; Nour-Eldin, Hussam Hassan; Halkier, Barbara Ann

    2016-01-01

    Oocytes of the African clawed frog, Xenopus laevis, are often used for expression and biochemical characterization of transporter proteins as the oocytes are particularly suitable for uptake assays and electrophysiological recordings. Assessment of the expression level of expressed transporters at the individual oocyte level is often desirable when comparing properties of wild type and mutant transporters. However, a large content of yolk platelets in the oocyte cytoplasm makes this a challenging task. Here we report a method for fast and easy, semiquantitative Western blot analysis of proteins heterologously expressed in Xenopus oocytes. PMID:26843169

  2. Developmental competence of oocytes grown in vitro: Has it peaked already?

    PubMed Central

    MOROHAKU, Kanako; HIRAO, Yuji; OBATA, Yayoi

    2015-01-01

    In vitro growth of immature oocytes provides opportunities to increase gametic resources and to understand the mechanisms underlying oocyte development. Many studies on the in vitro growth of oocytes have been reported thus far; however, only a few cases have been reported, which demonstrated that oocytes can support full-term development after in vitro fertilization. Our research group recently found that culture of mouse neonatal primordial follicles increased the birthrate; however, the establishment of an in vitro system that can completely mimic follicle or oocyte growth in vivo and control oogenesis remains an ongoing challenge. PMID:26685717

  3. Steroid hormones promote bovine oocyte growth and connection with granulosa cells.

    PubMed

    Makita, Miho; Miyano, Takashi

    2014-09-01

    Many approaches have been investigated for growing oocytes in vitro in mammals. To support oocyte growth in vitro, the culture systems must meet certain conditions for maintaining connections between oocytes and surrounding granulosa cells. The aims of this study were to determine the effects of combinations of 17β-estradiol (E2) and androstenedione (A4) on in vitro growth of bovine oocytes and to determine the number of connections between the oocyte and granulosa cells. Oocyte-granulosa cell complexes (OGCs) collected from early antral follicles (0.4-0.7 mm in diameter) were cultured for 14 days in a medium with different concentrations of E2 and A4, either alone or in combinations. We then assessed the number of transzonal projections (TZPs), which extend from granulosa cells through the zona pellucida to the oolemma. During in vitro growth culture, OGC structures were maintained in the medium with steroid hormones. The mean diameter of oocytes grown in the medium with both E2 and A4 was increased from 95.8 μm to around 120 μm, larger than oocytes grown without steroid hormones (109.9 μm) and similar in size to in vivo fully grown oocytes (119.4 μm) from 4- to 6-mm antral follicles. In subsequent in vitro maturation culture (22 hours), 30% (12 of 40) and 34% (14 of 41) of oocytes grown with E2 or A4 alone, respectively, matured to metaphase II; meanwhile, oocytes grown with a combination of E2 and A4 matured to metaphase II at a high rate (58%, 23 of 40). Growing oocytes isolated from early antral follicles had many uniformly distributed TZPs throughout the zona pellucida. After 14 days of culture, there was a significant decrease in the number of TZPs in oocytes grown without steroid hormones, whereas the number of TZPs was maintained in oocytes grown with steroid hormones. In particular, oocytes grown with E2 alone or with a combination of E2 and A4 had numbers of TZPs similar to oocytes before growth culture. In conclusion, a combination of E2 and A4 maintained the connections between oocytes and granulosa cells during in vitro growth culture of bovine oocytes for 14 days, resulting in the complete oocyte growth and the acquisition of meiotic competence in more than half the oocytes. PMID:24985562

  4. Chromosome Cohesion Established by Rec8-Cohesin in Fetal Oocytes Is Maintained without Detectable Turnover in Oocytes Arrested for Months in Mice.

    PubMed

    Burkhardt, Sabrina; Borsos, Máté; Szydlowska, Anna; Godwin, Jonathan; Williams, Suzannah A; Cohen, Paula E; Hirota, Takayuki; Saitou, Mitinori; Tachibana-Konwalski, Kikuë

    2016-03-01

    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 [2-6]. 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

  5. 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

  6. 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 genomics studies. PMID:25398096

  7. Chromatin configuration within the germinal vesicle of horse oocytes: changes post mortem and relationship to meiotic and developmental competence.

    PubMed

    Hinrichs, K; Choi, Y H; Love, L B; Varner, D D; Love, C C; Walckenaer, B E

    2005-05-01

    We evaluated the relationship of initial chromatin configuration to time of oocyte recovery and to nuclear maturation after culture in horse oocytes having compact (Cp) and expanded (Ex) cumuli. In addition, we evaluated the effect of oocyte type, time of recovery, and duration of culture on blastocyst development after intracytoplasmic sperm injection. In oocytes collected within 1 h of slaughter, fibrillar and intermediate chromatin configurations were more prevalent in Cp than in Ex oocytes (68% and 12%, respectively). In Cp oocytes collected after a 5- to 9-h delay, the proportions in the fibrillar and intermediate configurations decreased significantly, and the proportions of degenerating and homogeneously fluorescent configurations increased. When cultured, 20% of oocytes classified as having fibrillar chromatin resumed meiosis, whereas 82% of intermediate and 81% to 86% of condensed chromatin oocytes did so. Meiotic resumption was higher in oocytes recovered immediately after slaughter, but these oocytes took longer to mature. Duration of maturation significantly affected blastocyst development rates in Cp oocytes recovered after a delay (13% and 38% for oocytes matured 24 and 36 h, respectively). Oocytes recovered after a delay had higher blastocyst development rates than did those collected immediately after slaughter. We conclude that the fibrillar and intermediate chromatin configurations may degenerate during ovary storage, resulting in decreased maturation rates, especially of Cp oocytes. Time of oocyte recovery and duration of maturation significantly affect the rate of blastocyst development. Oocytes with Cp and Ex cumuli have similar developmental competence to the blastocyst stage. PMID:15647456

  8. Anastral spindle assembly and ?-tubulin in Drosophila oocytes

    PubMed Central

    2011-01-01

    Background Anastral spindles assemble by a mechanism that involves microtubule nucleation and growth from chromatin. It is still uncertain whether ?-tubulin, a microtubule nucleator essential for mitotic spindle assembly and maintenance, plays a role. Not only is the requirement for ?-tubulin to form anastral Drosophila oocyte meiosis I spindles controversial, but its presence in oocyte meiosis I spindles has not been demonstrated and is uncertain. Results We show, for the first time, using a bright GFP fusion protein and live imaging, that the Drosophila maternally-expressed ?Tub37C is present at low levels in oocyte meiosis I spindles. Despite this, we find that formation of bipolar meiosis I spindles does not require functional ?Tub37C, extending previous findings by others. Fluorescence photobleaching assays show rapid recovery of ?Tub37C in the meiosis I spindle, similar to the cytoplasm, indicating weak binding by ?Tub37C to spindles, and fits of a new, potentially more accurate model for fluorescence recovery yield kinetic parameters consistent with transient, diffusional binding. Conclusions The FRAP results, together with its mutant effects late in meiosis I, indicate that ?Tub37C may perform a role subsequent to metaphase I, rather than nucleating microtubules for meiosis I spindle formation. Weak binding to the meiosis I spindle could stabilize pre-existing microtubules or position ?-tubulin for function during meiosis II spindle assembly, which follows rapidly upon oocyte activation and completion of the meiosis I division. PMID:21208439

  9. 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.

  10. Characterization of 1-methyladenine binding in starfish oocyte cortices

    SciTech Connect

    Yoshikuni, Michiyasu Shizuoka Univ. ); Ishikawa, Katsutoshi ); Isobe, Minoru; Goto, Toshio ); Nagahama, Yoshitaka )

    1988-03-01

    1-Methyladenine (1MeAde) is the naturally occurring maturation-inducing hormone of starfish oocytes. The authors have prepared a biologically active ({sup 3}H)1MeAde of high purity and relatively high specific radioactivity. This ligand binds to cortices isolated from full-grown prophase-arrested oocytes of the starfish Asterina pectinifera. The binding of ({sup 3}H)1MeAde to cortices was rapid and reached equilibrium in {approx}15 min. This is in excellent agreement with the hormone-dependent period required for the induction of oocyte maturation. Binding was maximal between pH 6.4 and 8.0 and diminished sharply above and below this range. Analysis of Scatchard plots of the equilibrium binding of ({sup 3}H)1MeAde to cortices indicates the existence of a single class of binding site with a dissociation constant of 0.3 {mu}M and a binding capacity of 0.02 fmol per cortex. Whereas biologically active analogs (1-benzyladenine, 1-ethyladenine) inhibited the specific binding of ({sup 3}H)1MeAde by cortices, biologically inactive analogs (2-methyladenine, 3-methyladenine, 1,9-dimethyladenine, and 1-methylhypoxanthine) did not. These results suggest that the 1MeAde binding characterized herein is necessary for the maturational action of 1MeAde on starfish oocytes.

  11. Aurelia aurita (Cnidaria) Oocytes' Contact Plate Structure and Development

    PubMed Central

    Adonin, Leonid S.; Shaposhnikova, Tatyana G.; Podgornaya, Olga

    2012-01-01

    One of the A. aurita medusa main mesoglea polypeptides, mesoglein, has been described previously. Mesoglein belongs to ZP-domain protein family and therefore we focused on A.aurita oogenesis. Antibodies against mesoglein (AB RA47) stain the plate in the place where germinal epithelium contacts oocyte on the paraffin sections. According to its position, we named the structure found the contact plate. Our main instrument was AB against mesoglein. ZP-domain occupies about half of the whole amino acid sequence of the mesoglein. Immunoblot after SDS-PAGE and AU-PAGE reveals two charged and high Mr bands among the female gonad germinal epithelium polypeptides. One of the gonads' polypeptides Mr corresponds to that of mesogleal cells, the other ones' Mr is higher. The morphological description of contact plate formation is the subject of the current work. Two types of AB RA47 positive granules were observed during progressive oogenesis stages. Granules form the contact plate in mature oocyte. Contact plate of A.aurita oocyte marks its animal pole and resembles Zona Pellucida by the following features: (1) it attracts spermatozoids; (2) the material of the contact plate is synthesized by oocyte and stored in granules; (3) these granules and the contact plate itself contain ZP domain protein(s); (4) contact plate is an extracellular structure made up of fiber bundles similar to those of conventional Zona Pellucida. PMID:23185235

  12. The portrayal of healthy women requesting oocyte cryo-preservation.

    PubMed

    Mertes, H

    2013-01-01

    The possibility to cryopreserve oocytes to be used in IVF treatment later in life has not only enlarged the reproductive options of cancer patients who are faced with gonadotoxic treatments, but also holds the promise of enlarging the reproductive options of healthy women whose personal circumstances (most often the absence of a partner) do not allow them to reproduce in their most fertile years. Opinions for and against this application of the cryopreservation technology are often based on different portrayals of the women who might use it. Three different portrayals can be discerned in the debate about the ethics of so-called 'social egg freezing' or 'non medical egg freezing'. First, these women have been portrayed as selfish career-pursuing women. Second, healthy women who might benefit from oocyte cryopreservation have been portrayed as victims of a male-oriented society that makes it difficult for women to combine motherhood with a good education or professional responsibilities. Third, healthy women -opting to cryopreserve oocytes have been portrayed as wise, proactive women who will not have to depend on -oocyte donors should they suffer from age-related infertility by the time they are ready to reproduce. Each of these three portrayals has its own shortcomings that one should be wary of, as they lead to an oversimplification of the ethical debate. PMID:24753939

  13. 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. PMID:24219576

  14. Centrifugation of bovine oocytes for nuclear micromanipulation and sperm microinjection.

    PubMed

    Tatham, B G; Sathananthan, A H; Dharmawardena, V; Munesinghe, D Y; Lewis, I; Trounson, A O

    1996-07-01

    The reproductive biotechnologies of intracytoplasmic sperm microinjection, nuclear transfer and DNA microinjection require the visualization of cytoplasmic components and nuclei of oocytes and early embryonic cells. Bovine oocytes were matured and fertilized in vitro, and then centrifuged at the germinal vesicle, metaphase II and pronuclear stages and at syngamy. These (n = 536) were examined using light and transmission electron microscopy. The organelles stratified in five distinct zones in a consistent pattern in both oocytes and zygotes, though relative fractions changed in organelle composition after fertilization. These comprised a centripetal lipid zone, below which was a vesicular zone, then a supra-equatorial band of smooth endoplasmic reticulum (SER), a clear zone and a centrifugal mitochondrial zone. Cortical granules were located peripherally, single or clumped together, in the clear and mitochondrial zones. The nuclei were usually found associated with the SER or Golgi membranes, and chromatin was clumped at one pole within the nucleus. The maturation spindles were often located beneath the oolemma in all zones, while the first mitotic spindle was usually located in the clear zone. Some of the oocytes were activated by centrifugation and completed maturation. PMID:8671492

  15. 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

  16. Fourteen babies born after round spermatid injection into human oocytes.

    PubMed

    Tanaka, Atsushi; Nagayoshi, Motoi; Takemoto, Youichi; Tanaka, Izumi; Kusunoki, Hiroshi; Watanabe, Seiji; Kuroda, Keiji; Takeda, Satoru; Ito, Masahiko; Yanagimachi, Ryuzo

    2015-11-24

    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

  17. 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

  18. The portrayal of healthy women requesting oocyte cryopreservation

    PubMed Central

    Mertes, H.

    2013-01-01

    The possibility to cryopreserve oocytes to be used in IVF treatment later in life has not only enlarged the reproductive options of cancer patients who are faced with gonadotoxic treatments, but also holds the promise of enlarging the reproductive options of healthy women whose personal circumstances (most often the absence of a partner) do not allow them to reproduce in their most fertile years. Opinions for and against this application of the cryopreservation technology are often based on different portrayals of the women who might use it. Three different portrayals can be discerned in the debate about the ethics of so-called social egg freezing or non medical egg freezing. First, these women have been portrayed as selfish career-pursuing women. Second, healthy women who might benefit from oocyte cryopreservation have been portrayed as victims of a male-oriented society that makes it difficult for women to combine motherhood with a good education or professional responsibilities. Third, healthy women opting to cryopreserve oocytes have been portrayed as wise, proactive women who will not have to depend on oocyte donors should they suffer from age-related infertility by the time they are ready to reproduce. Each of these three portrayals has its own shortcomings that one should be wary of, as they lead to an oversimplification of the ethical debate. PMID:24753939

  19. Regimen of ovarian stimulation affects oocyte and therefore embryo quality.

    PubMed

    Bosch, Ernesto; Labarta, Elena; Kolibianakis, Efstratios; Rosen, Mitchell; Meldrum, David

    2016-03-01

    Without any doubt the regimen used to mature multiple capable oocytes for IVF impacts IVF outcomes. Studies have indicated that the inclusion of LH activity, adjuvant agents such as growth hormone (GH), and regimens providing for simultaneous action of both LH and FSH during final oocyte maturation may have beneficial effects on IVF outcomes. Because of the difficulty in improving IVF outcomes in poor responders, the studies on GH are of particular interest. As pointed out in this review, the apparent beneficial effects of GH on oocyte competence may also apply to older women or to normal responders with reduced embryo quality. A much more difficult question is whether and how much ovarian stimulation impacts on oocyte competence. Paradoxically it seems that there are not demonstrated differences between the stimulated and the natural unstimulated cycle, whereas studies in laboratory animals and IVF patients have shown deleterious effects of higher compared with lower doses of gonadotropins. Recent studies suggest that the use of high doses of gonadotropins as an independent factor correlates negatively with the probability of live birth, whereas a high ovarian response per se is associated with better cumulative pregnancy rates, owing to the availability of more euploid and good-quality embryos. Although adjunctive use of androgens has not been discussed here, it is briefly covered in the first review of this series. PMID:26826273

  20. 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.

  1. 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. PMID:16569309

  2. Raman Micro-Spectroscopy Can Be Used to Investigate the Developmental Stage of the Mouse Oocyte

    PubMed Central

    Davidson, Bryony; Murray, Alison A.

    2013-01-01

    In recent years, the uptake of assisted reproductive techniques such as in vitro fertilisation has risen exponentially. However, there is much that is still not fully understood about the biochemical modifications that take place during the development and maturation of the oocyte. As such, it is essential to further the understanding of how oocyte manipulation during these procedures ultimately affects its developmental potential; yet, there are few methods currently available which are capable of providing a quantitative measure of oocyte quality. Raman spectroscopy enables investigation of the global biochemical profile of intact cells without the need for labelling. Here, Raman spectra were acquired from the ooplasm of mouse oocytes at various stages of development, from late pre-antral follicles, collected after in vitro maturation within their ovarian follicles and from unstimulated and stimulated ovulatory cycles. Using a combination of univariate and multivariate statistical methods, it was found that ooplasm lipid content could be used to discriminate between different stages of oocyte development. Furthermore, the spectral profiles of mature oocytes revealed that oocytes which have developed in vitro are protein-deficient when compared to in vivo grown oocytes. Finally, the ratio of two Raman peak intensities, namely 1605?1447 cm?1, used as a proxy for the protein-to-lipid ratio of the ooplasm, was shown to be indicative of the oocytes quality. Together, results indicate that Raman spectroscopy may present an alternative analytical tool for investigating the biochemistry of oocyte developmental stage and quality. PMID:23840882

  3. Thioredoxin-interacting protein regulates glucose metabolism and affects cytoplasmic streaming in mouse oocytes.

    PubMed

    Lee, Su-Yeon; Lee, Hyun-Seo; Kim, Eun-Young; Ko, Jung-Jae; Yoon, Tae Ki; Lee, Woo-Sik; Lee, Kyung-Ah

    2013-01-01

    Thioredoxin-interacting protein (Txnip) regulates intracellular redox state and prompts oxidative stress by binding to and inhibiting Thioredoxin (Trx). In addition, via a Trx-independent mechanism, Txnip regulates glucose metabolism and thus maintains intracellular glucose levels. Previously, we found Txnip mRNA highly expressed in immature germinal vesicle (GV) oocytes, but currently there is no report describing the role of Txnip in oocytes. Therefore, we conducted the present study to determine the function of Txnip in mouse oocytes' maturation and meiosis by using RNA interference (RNAi) method. Upon specific depletion of Txnip, 79.5% of oocytes were arrested at metaphase I (MI) stage. Time-lapse video microscopy analysis revealed that the formation of granules in the oocyte cytoplasm increased concurrent with retarded cytoplasmic streaming after Txnip RNAi treatment. Txnip RNAi-treated oocytes had upregulated glucose uptake and lactate production. To confirm the supposition that mechanism responsible for these observed phenomena involves increased lactate in oocytes, we cultured oocytes in high lactate medium and observed the same increased granule formation and retarded cytoplasmic streaming as found by Txnip RNAi. The MI-arrested oocytes exhibited scattered microtubules and aggregated chromosomes indicating that actin networking was disturbed by Txnip RNAi. Therefore, we conclude that Txnip is a critical regulator of glucose metabolism in oocytes and is involved in maintaining cytoplasmic streaming in mouse oocytes. PMID:23976953

  4. Gene Expression Profiling of Human Oocytes Developed and Matured In Vivo or In Vitro

    PubMed Central

    Virant-Klun, Irma; Knez, Katja; Tomazevic, Tomaz; Skutella, Thomas

    2013-01-01

    The quality of the human oocyte determines the success of fertilization and affects the consequent embryo development, pregnancy and birth; it therefore serves as a basis for human reproduction and fertility. The possibility to evaluate oocyte quality in the in vitro fertilization programme is very limited. The only criterion which is commonly used to evaluate oocyte quality is its morphology. There is a mass of oocytes in the in vitro fertilization programme which are not fertilized in spite of normal morphology. In the past, several attempts focused on oocyte gene expression profiling by different approaches. The results elucidated groups of genes related to the human oocyte. It was confirmed that some factors, such as oocyte in vitro maturation, are detectable at the molecular level of human oocytes and their polar bodies in terms of gene expression profile. Furthermore, the first genetic evaluations of oocyte-like cells developed in vitro from human stem cells of different origin were performed showing that these cells express some genes related to oocytes. All these findings provide some new knowledge and clearer insights into oocyte quality and oogenesis that might be introduced into clinical practice in the future. PMID:23509795

  5. Nucleo-cytoplasmic interactions in cell hybrids between mouse oocytes, blastomeres and somatic cells.

    PubMed

    Tarkowski, A K; Ba?akier, H

    1980-02-01

    With the help of the technique of Sendai virus-mediated cell fusion, hybrid cells were produced between two maturing oocytes, between maturing oocytes or mature secondary oocytes and interphase blastomeres from 2-cell embryos, and between secondary oocytes and follicle cells (FC). In the first case giant oocytes form and in these the two groups of condensing bivalents join on a common spindle, undergo a first meiotic division, and become arrested in metaphase II; these hybrids are heterozygous diploids. When blastomeres are fused with oocytes undergoing maturation, then the blastomere nucleus undergoes premature chromosome condensation (PCC), and two sets of chromosomes (meiotic and mitotic) come close to each other but the mitotic chromosomes are not fully incorporated into the meiotic spindle. The behaviour of the nuclei from blastomeres or from follicle cells fused with secondary ovulated oocytes, depends on whether or not the oocyte undergoes activation. When the oocyte is not activated, then the introduced nuclei undergo PCC but the chromosomes remain separate from the spindle of metaphase II. When the oocyte is activated, then the introduced nuclei remain in interphase; the FC nuclei may increase their volume eight times and undergo some other structural changes but during the 6 h period after fusion their appearance remains distinct from that of the female pronucleus. Since some oocytes are not activated after fusion has occurred, it follows that the fusion process itself is not sufficient to trigger egg activation. PMID:7373200

  6. The effects of voluntary exercise on oocyte quality in a diet-induced obese murine model.

    PubMed

    Boudoures, Anna L; Chi, Maggie; Thompson, Alysha; Zhang, Wendy; Moley, Kelle H

    2016-03-01

    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 that dietary interventions fail to reverse these problems. How exercise affects the oocytes has not been addressed. Therefore, we hypothesized an exercise intervention would improve oocyte quality. Here we show that 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 hydroxyacyl-coenzyme A 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 an HFD and obesity. By utilizing an exercise intervention on an 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

  7. 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

  8. Translocation and Endocytosis for Cell-penetrating Peptide Internalization

    PubMed Central

    Jiao, Chen-Yu; Delaroche, Diane; Burlina, Fabienne; Alves, Isabel D.; Chassaing, Grard; 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

  9. Embryo apoptosis identification: Oocyte grade or cleavage stage?

    PubMed

    Bakri, Noraina Mohd; Ibrahim, Siti Fatimah; Osman, Nurul Atikah; Hasan, Nurhaslina; Jaffar, Farah Hanan Fathihah; Rahman, Zulaiha Abdul; Osman, Khairul

    2016-01-01

    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

  10. 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

  11. Full-grown oocytes from Xenopus laevis resume growth when placed in culture

    SciTech Connect

    Wallace, R.A.; Misulovin, Z.; Etkin, L.D.

    1981-05-01

    When most full-grown, follicle cell-invested oocytes from Xenopus laevis are placed in an appropriate culture medium, they resume growth and remain physiologically healthy for at least 2 to 3 weeks. Rates of growth by full-grown oocytes in vitro generally approximate and can even exceed the most rapid growth rate achieved by vitellogenic oocytes in vivo. Resumption of oocyte growth can be correlated with the loss of investing follicle cells, which under normal conditions appear to interfere with vitellogenin and nutrient access to the oocyte. The final size reached by the oocyte within the ovary is thus not an intrinsic property of the oocyte but is extrinsically imposed by the somatic environment.

  12. Calcium transients during early development in single starfish (Asterias forbesi) oocytes

    SciTech Connect

    Eisen, A.; Reynolds, G.T.

    1984-11-01

    Maturation and fertilization of the starfish oocyte are putative calcium-dependent events. The authors have investigated the spatial distribution and temporal dynamics of this calcium dependence in single oocytes of Asterias forbesi. They used the calcium photoprotein, aequorin, in conjunction with a microscope-photomultiplier and microscope-image intensifier. Surprisingly, in contrast to earlier work with Marasthenias glacialis, there is no detectable increase in intracellular-free calcium in the oocyte of A. forbesi in response to the maturation hormone 1-methyl adenine. During fertilization of the same, matured, A. forbesi oocyte there is a large increase in intracellular-free calcium. The calcium concentration increases to approx.1 ..mu..M at the point of insemination and the region of elevated free calcium expands across the oocyte in approx.20 s (17-19/sup 0/C). After the entire oocyte reaches an elevated concentration of free calcium, the concentration decreases uniformly throughout the oocyte over the next several minutes.

  13. 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.

  14. Femtosecond laser based enucleation of porcine oocytes for somatic cell nuclear transfer

    NASA Astrophysics Data System (ADS)

    Ktemeyer, 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.

  15. 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.

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

    PubMed

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

    2014-12-01

    Glycoprotein oligosaccharides play major roles during reproduction, yet their function in gamete interactions is not fully elucidated. Identification and comparison of the glycan pattern in cumulus-oocyte complexes (COCs) from species with different efficiencies of in vitro spermatozoa penetration through the zona pellucida (ZP) could help clarify how oligosaccharides affect gamete interactions. We compared the expression and localization of 12 glycosidic residues in equine and porcine in vitro-matured (IVM) and preovulatory COCs by means of lectin histochemistry. The COCs glycan pattern differed between animals and COC source (IVM versus preovulatory). Among the 12 carbohydrate residues investigated, the IVM COCs from these two species shared: (a) sialo- and ?N-acetylgalactosamine (GalNAc)-terminating glycans in the ZP; (b) sialylated and fucosylated glycans in cumulus cells; and (c) GalNAc and N-acetylglucosamine (GlcNAc) glycans in the ooplasm. Differences in the preovulatory COCs of the two species included: (a) sialoglycans and GlcNAc terminating glycans in the equine ZP versus terminal GalNAc and internal GlcNAc in the porcine ZP; (b) terminal galactosides in equine cumulus cells versus terminal GlcNAc and fucose in porcine cohorts; and (c) fucose in the mare ooplasm versus lactosamine and internal GlcNAc in porcine oocyte cytoplasm. Furthermore, equine and porcine cumulus cells and oocytes contributed differently to the synthesis of ZP glycoproteins. These results could be attributed to the different in vitro fertilization efficiencies between these two divergent, large-animal models. PMID:25511183

  17. 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.

  18. Uranium in drinking water: effects on mouse oocyte quality.

    PubMed

    Kundt, Miriam S; Martinez-Taibo, Carolina; Muhlmann, Maria C; Furnari, Juan C

    2009-05-01

    The aim of this work was to evaluate the reproductive toxicological effects of uranium (U) at 2.5, 5, and 10 mgU/kg/d chronically administered in drinking water for 40 d. Swiss female control mice (n = 28) and mice chronically contaminated with uranyl nitrate in drinking water (n = 36) were tested. The number and quality of ovulated oocytes, chromatin organization, and nuclear integrity were evaluated. No significant differences were obtained in the numbers of ovulated oocytes between the different groups. Nevertheless, in 1,520 of the oocytes examined, dysmorphism increased from 11.99% in the control group to 27.99%, 27.19%, and 27.43% in each of the contaminated groups, respectively, in a dose-independent manner. On the other hand, morphological chromatin organization from 880 oocytes examined showed an increase in metaphase plate abnormalities from 37.20% (+/-7.21) in the control group to 55.13% (+/-21.36), 58.29% (+/-21.72), and 64.10% (+/-12.62) in each of the contaminated groups, respectively. Cumulus cell (CC) micronucleation, a parameter of nuclear integrity, increased from 0.21% (+/-0.31) in the control group to 1.92 (+/-0.95), 2.98 (+/-0.97), and 3.2 (+/-0.98), respectively. Both metaphase plate abnormalities and CC micronucleation showed an increase in a dose-dependent manner (r = 0.9; p < 0.001). The oocyte and its microenvironment showed high sensitivity to uranium contamination by drinking water. The lowest observed adverse effect level for this system is estimated at a level below 2.5 mgU/kg/d for female mice. PMID:19359850

  19. Milder Etchant For Penetrant Inspection

    NASA Technical Reports Server (NTRS)

    O'Tousa, Joseph E.; Thomas, Clark S.

    1990-01-01

    New etching solution for chemical penetrant inspection of Inconel(R) 718 castings and weldments. Etchant does not introduce artifacts mistaken for defects. Applied by swabbing or by immersion. Used to detect unwanted residues of Nioro(R) (or equivalent) gold brazing alloy on type 347 stainless steel.

  20. 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.

  1. 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.

  2. Effect of androstenedione on the growth and meiotic competence of bovine oocytes from early antral follicles.

    PubMed

    Taketsuru, Hiroaki; Hirao, Yuji; Takenouchi, Naoki; Iga, Kosuke; Miyano, Takashi

    2012-11-01

    Medium that contains 17β-estradiol has been reported to support in vitro growth of bovine oocytes, isolated from early antral follicles, until the final stage. The aim of this study was to determine the effects of androstenedione in medium on such growing bovine oocytes. Oocyte-granulosa cell complexes were collected from early antral follicles and cultured for 14 days in medium supplemented with 17β-estradiol (0, 10 and 100 ng/ml) or androstenedione (0, 10 and 100 ng/ml). The mean diameter of oocytes measured after seeding on the culture substrate was 96.9 μm (n = 191). Either steroid was necessary for maintainance of the organization of oocyte-granulosa cell complexes over the 14-day culture period. In the 17β-estradiol- or the androstenedione-supplemented medium about 80% or 65%, respectively, of viable oocytes were recovered. In both groups the increase in oocyte size was significant after 14 days. The in vitro grown oocytes were cultured for a further 22-24 h for oocyte maturation; 13% and 30% of oocytes grown in the 10 and 100 ng/ml 17β-estradiol-supplemented medium reached metaphase II, respectively; more than 64% of oocytes grown in the androstenedione-supplemented medium matured to metaphase II. These results show that androstenedione, as 17β-estradiol, can maintain the viability of bovine oocyte-granulosa cell complexes and support the growth of oocytes, and that androstenedione promotes the acquisition of oocyte meiotic competence efficiently at a low dose. PMID:22067704

  3. The quality of porcine oocytes is affected by sexual maturity of the donor gilt.

    PubMed

    Pawlak, Piotr; Renska, Natalia; Pers-Kamczyc, Emilia; Warzych, Ewelina; Lechniak, Dorota

    2011-03-01

    Although differences in the quality of oocytes derived from young gilts and adult sows are well documented, evidence concerning gametes of pre-pubertal and cycling gilts is scarce and inconsistent. The aim of this work was to establish whether sexual maturity of gilts affects the quality of their oocytes with the use of the brilliant cresyl blue (BCB) test, oocyte diameter and apoptosis. Ovarian morphology was evaluated, and gonads with corpus luteum or albicans were recognized as originating form cycling gilts (C) and those with follicles as originating form pre-pubertal females (P). Altogether 952 cumulus-oocyte complexes (COCs; group P: 554; group C: 398) were examined, whereas 149 COCs, not subjected to BCB test, served as a control for TUNEL. COCs of proper morphology were evaluated by the BCB test which differentiated two categories of gametes: more competent, BCB+, and less competent BCB- oocytes. The control group comprised oocytes of proper morphology aspirated from ovaries of P and C gilts not subjected to BCB test. Finally five groups of COCs were matured in vitro: 1/P-BCB+, 2/P-BCB-, 3/C-BCB+, 4/ C-BCB- and 5/ control. Significantly more large oocytes (? 120 m), more BCB+ oocytes and more high quality (both BCB+ and ? 120 m) oocytes originated from ovaries of cycling gilts than pre-pubertal gilts (p<0.001). The rate of mature oocytes at the MII stage differed significantly between C-BCB+ (68.5%) and P-BCB+ (32.9%) oocytes. The incidence of apoptosis among BCB-treated oocytes after in vitro maturation was 21.4% and was similar to that observed in control oocytes (17.4%). BCB+ oocytes from cycling gilts showed significantly higher (28.7%) incidence of apoptosis than that of the group P (16.2%). Interestingly, high quality oocytes displayed a similar level of apoptosis regardless of the donor puberty. We demonstrated that C gilts provided more BCB+ oocytes as well as more large oocytes than P gilts, although C-BCB+ oocytes showed higher apoptotic rate. In conclusion, high incidence of apoptosis and a big variation in the diameter of more competent BCB+ oocytes make the BCB test a less effective selection tool than previously reported. PMID:21455277

  4. 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. PMID:18210127

  5. DNA SYNTHESIS IN THE FERTILIZING HAMSTER SPERM NUCLEUS: SPERM TEMPLATE AVAILABILITY AND EGG CYTOPLASMIC CONTROL

    EPA Science Inventory

    To assess the role of sperm template availability in the regulation of DNA synthesis, the morphological status of the fertilizing hamster sperm nucleus was correlated with its ability to synthesize DNA after in vivo and in vitro fertilization. Fertilized hamster eggs were incubat...

  6. A Comparison of Hamster Anesthetics and Their Effect on Mosquito Blood Feeding

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Hamsters or mice are often anesthetized when they are used as the hosts for insect feeding experiments. An experiment was done to determine if there was a difference in mosquito blood feeding success when fed on hamsters anesthetized using two commonly used protocols. The number of blood-fed females...

  7. Corn fiber oil and sitostanol decrease cholesterol absorption independently of intestinal sterol transporters in hamsters

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The aim of this study was to investigate the cholesterol-lowering mechanism of corn fiber oil (CFO), ferulate phytostanyl esters (FPE) and parent compounds including sitostanol and ferulic acid in hamsters. Method: Seventy male golden syrian hamsters were randomly assigned to six experimental diets ...

  8. 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

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

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 9 Animals and Animal Products 1 2011-01-01 2011-01-01 false Primary enclosures used to transport... enclosures used to transport live guinea pigs and hamsters. No person subject to the Animal Welfare regulations shall offer for transportation, or transport, in commerce any live guinea pig or hamster in...

  10. 9 CFR 3.36 - Primary enclosures used to transport live guinea pigs and hamsters.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Primary enclosures used to transport... enclosures used to transport live guinea pigs and hamsters. No person subject to the Animal Welfare regulations shall offer for transportation, or transport, in commerce any live guinea pig or hamster in...

  11. 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. PMID:21889598

  12. 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

  13. BODY TEMPERATURE IN THE MOUSE, HAMSTER, AND RAT EXPOSED TO RADIOFREQUENCY RADIATION: AN INTERSPECIES COMPARISON

    EPA Science Inventory

    Colonic temperatures of BALB/c and CBA/J mice, golden hamsters, and Sprague-Dawley rats were taken immediately after exposure for 90 min to radiofrequency (RF) radiation. Exposures were made in 2450 MHz (mouse and hamster) or 600 MHz (rat) waveguide exposure systems while the dos...

  14. OBSERVATIONS OF SYRIAN HAMSTER FETUSES AFTER EXPOSURE TO 2450-MHZ MICROWAVES

    EPA Science Inventory

    The teratogenic potential of microwaves was examined in a rodent species, the Syrian hamster. Exposure of hamsters to 2450-MHz CW microwaves at a power denisty of 20 mW/sq. cm. for 100 minutes daily on days 6-14 of gestation caused no significant change in fetal survival, body we...

  15. 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. PMID:26674551

  16. 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).

  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. Penetration below a convective zone

    NASA Technical Reports Server (NTRS)

    Hurlburt, Neal E.; Toomre, Juri; Massaguer, Josep M.; Zahn, Jean-Paul

    1994-01-01

    Two-dimensional numerical simulations are used to investigate how fully compressible nonlinear convection penetrates into a stably stratified zone beneath a stellar convection zone. Estimates are obtained of the extent of penetration as the relative stability S of the stable to the unstable zone is varied over a broad range. The model deals with a perfect gas possessing a constant dynamic viscosity. The dynamics is dominated by downward-directed plumes which can extend far into the stable material and which can lead to the excitation of a broad spectrum of internal gravity waves in the lower stable zone. The convection is highly time dependent, with the close coupling between the lateral swaying of the plumes and the internal gravity waves they generate serving to modulate the strength of the convection. The depth of penetration delta, determined by the position where the time-averaged kinetic flux has its first zero in the stable layer, is controlled by a balance between the kinetic energy carried into the stable layer by the plumes and the buoyancy braking they experience there. A passive scalar is introduced into the unstable layer to evaluate the transport of chemical species downward. Such a tracer is effectively mixed within a few convective overturning times down to a depth of delta within the stable layer. Analytical estimates based on simple scaling laws are used to interpret the variation of delta with S, showing that it first involves an interval of adiabatic penetration if the local Peclet number of the convection exceeds unity, followed by a further thermal adjustment layer, the depths of each interval scaling in turn as S(exp -1) and S(exp -1/4). These estimates are in accord with the penetration results from the simulations.

  19. 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. PMID:24909854

  20. Aquaporin-11 Control of Testicular Fertility Markers in Syrian Hamsters

    PubMed Central

    Shannonhouse, John L.; Urbanski, Henryk F.; Woo, Shih-Lung; Fong, Li An; Goddard, Scott D.; Lucas, William F.; Jones, Edward R.; Wu, Chaodong; Morgan, Caurnel

    2015-01-01

    The present study sought novel changes to the hamster testicular transcriptome during modulation of fertility by well-characterized photoperiodic stimuli. Transition from long days (LD, 14 h light/day) to short days (SD, 10 h light/day) triggered testicular regression (61% reduction of testis weight, relative to LD) in SD-sensitive (SD-S) hamsters within 16 weeks. After 22 weeks of SD exposure, a third cohort of hamsters became SD-refractory (SD-R), and exhibited testicular recrudescence (137% testis weight gain, relative to SD-S). Partial interrogation of the testicular transcriptome by annealing-control-primer-modified differential display PCR provided several candidates for regulation of testicular functions. Multiple linear regression modeling indicated the best correlation for aquaporin 11 (Aqp11) with changes in testis weight. Correlations were also strongest for Aqp11 with expression levels of reference cDNAs that control spermatogenesis (Hspa2 and Tnp2), steroidogenesis (Cox2, 3?Hsd, and Srebp2), sperm motility (Catsper1, Pgk2, and Tnp2), inflammation (Cox2), and apoptosis (Bax and Bcl2). Moreover, siRNA-mediated knockdown of testicular Aqp11 mRNA and protein reduced Hspa2 and Tnp2 mRNA levels, and it increased 3?Hsd mRNA levels. It also reduced mRNA levels for Sept12, which is a testis-specific inducer of spermatogenesis. These results suggest a central role for testicular Aqp11 signaling in the coordinate regulation of crucial components of fertility. PMID:24791736

  1. The circadian system of the Turkish hamster, Mesocricetus brandti: responses to light.

    PubMed

    Pohl, H

    1985-01-01

    The circadian system of the Turkish hamster controlling wheel-running activity responded to single 1-hr light pulses and to repeated 1-hr pulses in a similar way as that of Syrian hamsters studied previously. At constant light of 100 lx, the period length (tau) of the freerunning activity rhythm of Turkish hamsters was longer and the activity time (alpha) was shorter than that of Syrian hamsters. Among individuals, the ability of the system to be entrained by one 1-hr light pulse per cycle was related to the range (advance plus delay amplitude) of the phase response curve (PRC) derived from single light pulses and to the compression of alpha caused by the pulse Zeitgeber. The data support the hypothesis derived from experiments on Syrian hamsters that the range of the PRC is functionally related with alpha, possibly reflecting the phase relations (coupling) between two oscillators. PMID:2863051

  2. 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 procedure compared with aspiration procedures. For in vitro maturation of oocytes, TCM is superior to CR1 and CMRL as basic maturation medium for this species. PMID:15910927

  3. Conditioned defeat in the Syrian golden hamster (Mesocricetus auratus).

    PubMed

    Potegal, M; Huhman, K; Moore, T; Meyerhoff, J

    1993-09-01

    When singly housed under laboratory conditions, male Syrian golden hamsters routinely attack novel conspecific intruders introduced into their home cages. As we report here, after being repeatedly defeated by a larger, more aggressive intruder, such normal territorial aggression on the part of the resident hamsters is replaced by defensive behavior and flight. We have found that such conditioned defeat (CD) can be reliably induced by a series of 5-min trials with an aggressive intruder whether these trials are spread over 4 days or are all given on the same day. A useful behavioral criterion for the appearance of CD during acquisition is the first occurrence of anticipatory flight (AF), i.e., the first time the resident flees from the next aggressive intruder before being attacked. CD shows generalization: Animals trained to the AF criterion (AF Group) subsequently show defensive behavior toward, and even flee from, intruders which show absolutely no sign of aggressiveness. Animals in the AF Group persisted in such defense behavior for two test sessions; animals given three additional defeat trials beyond the appearance of AF (AF + 3 Group) showed a greater magnitude and persistence of defense and flight. A comparison of CD-trained animals which met a non-aggressive intruder (NAI) every day for 5 days to similarly trained animals which met the intruder only on the fifth day after acquisition suggests that CD diminishes passively as a function of time and not as the consequence of repeated encounters with a nonaggressive stimulus animal. We also found that near ideal NAIs could be prepared by treating nonaggressive hamsters with high doses of diazepam: animals so treated locomote more or less continuously around the cage virtually ignoring the subject. An unexpected observation was that subjects in the AF Group tended to closely follow these diazepam-treated, rapidly locomoting NAIs around the cage. Following may be an example of the "risk assessment" activities directed toward a potential threat. The development of a rapid and reliable technique for inducing CD in hamsters sets the stage for further physiological and pharmacological work on this interesting phenomenon. PMID:8117243

  4. 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}.

  5. 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.

  6. 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

  7. Both diet and gene mutation induced obesity affect oocyte quality in mice.

    PubMed

    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

  8. 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

  9. Effect of Vitamin E on Oocytes Apoptosis in Nicotine-Treated Mice

    PubMed Central

    Asadi, Ebrahim; Jahanshahi, Mehrdad; Golalipour, Mohammad Jafar

    2012-01-01

    Objective(s) Cigarette and nicotine enhances embryogenesis, fertility, pregnancy loss and ultrastructure alterations of oocyte. This study was performed to determine the effect of daily supplementation of vitamin E on oocytes apoptosis in nicotine-treated mice. Materials and Methods In this experimental study, 24 NMARI adult female mice were randomly allocated into four experimental groups. For 30 days, animals in control group (C) were received saline through subcutaneous injection, group I received vitamin E (60 mg/kg/day orally), group II received nicotine (5 mg/kg/day, subcutaneous) and animals of group III received nicotine with vitamin E (60 mg/kg/day orally). After 30 days, the animals were superovulated with PSMG (10 Units) and HCG (10 Units). Next day animals were sacrificed and oocytes were flushed. Collected oocytes were examined through TUNEL assay for the determination of apoptosis through the use of fluorescent microscope. Results The number of retrieved oocytes was 139, 148, 97 and 127 in control, experimental group I, II and III, respectively. Nicotine treatment increased apoptosis in oocytes up to 13.4% whereas oocytes apoptosis was 3.6% in controls. Supplementation with vitamin E in nicotine-treated mice reduced the oocytes apoptosis to 5.5%. Conclusion This study showed that nicotine exposure (5 mg/kg/day for 30 days) can increase apoptosis in oocytes, and supplementation with vitamin E (60 mg/kg/day orally) can reduce the oocytes apoptosis in nicotine-treated mice. PMID:23493325

  10. Isolation of ovarian components essential for growth and development of mammalian oocytes in vitro.

    PubMed

    Hirao, Yuji

    2012-01-01

    Mammalian ovaries contain a large number of oocytes, most of which degenerate either before or at various stages of growth. Dynamic and precise regulation in the ovary involves many factors, each with a unique role. Identifying the single most important factor is impossible; however, it may be possible to identify factors essential for oocyte growth. It is evident that oocytes can grow into competent ova in vitro; however, how faithfully the follicle should mimic the in vivo conditions remains unclear. In the culture system discussed in this review, bovine and mouse oocyte-granulosa cell complexes, at approximately the late mid-growth stage, spread on a substratum without the involvement of theca cells. The structural simplicity of this system is advantageous because it reduces the basic conditions essential for regulation of oocyte growth. Apart from biological factors, high concentrations of polyvinylpyrrolidone (molecular weight: 360000) improved oocyte growth. Among ovarian factors, androstenedione was used to compensate for the absence of theca cells, and it promoted both follicular growth and acquisition of oocyte meiotic competence. Most oocytes cultured in a group were viable after long-term culture, suggesting that unlike ovarian events, there was no exhaustive follicle selection. Collectively, oocytes and their associated granulosa cells can establish independent units capable of supporting oocyte growth in appropriately modified culture media. PMID:22738899

  11. 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

  12. Cumulus cells accelerate oocyte aging by releasing soluble Fas Ligand in mice

    PubMed Central

    Zhu, Jiang; Zhang, Jie; Li, Hong; Wang, Tian-Yang; Zhang, Chuan-Xin; Luo, Ming-Jiu; Tan, Jing-He

    2015-01-01

    Although previous studies have suggested that cumulus cells (CCs) accelerate oocyte aging by secreting soluble and heat-sensitive paracrine factors, the factors involved are not well characterized. Because Fas-mediated apoptosis represents a major pathway in induction of apoptosis in various cells, we proposed that CCs facilitate oocyte aging by releasing soluble Fas ligand (sFasL). In this study, we reported that when the aging of freshly ovulated mouse oocytes were studied in vitro, both the apoptotic rates of CCs and the amount of CCs produced sFasL increased significantly with the culture time. We found that oocytes expressed stable levels of Fas receptors up to 24?h of in vitro aging. Moreover, culture of cumulus-denuded oocytes in CCs-conditioned CZB medium (CM), in CZB supplemented with recombinant sFasL, or in CM containing sFasL neutralizing antibodies all showed that sFasL impaired the developmental potential of the oocytes whereas facilitating activation and fragmentation of aging oocytes. Furthermore, CCs from the FasL-defective gld mice did not accelerate oocyte aging due to the lack of functional FasL. In conclusion, we propose that CCs surrounding aging oocytes released sFasL in an apoptosis-related manner, and the released sFasL accelerated oocyte aging by binding to Fas receptors. PMID:25731893

  13. The study of mammalian oocyte competence by transcriptome analysis: progress and challenges.

    PubMed

    Labrecque, Rmi; Sirard, Marc-Andr

    2014-02-01

    Various morphological and cytological traits of oocytes and their surrounding cumulus cells may be used to select oocytes for assisted reproduction. However, even with careful selection, successful IVF and subsequent embryo development remain uncertain. The factors that ensure oocyte competence are unclear and other approaches to assessing developmental potential must be explored. With the constant development of the molecular toolbox, genomic/transcriptomic analysis is becoming a more and more interesting approach to understand oocyte quality on the basis of RNA composition. Using bovine and mouse models as well as human oocytes of known developmental potential, various efforts are underway to characterize the mRNA profile of the competent oocyte using microarray technology. The proliferation of gene expression data sets raises new opportunities to identify the mechanisms involved in this complex phenotype, which should lead to improved techniques of assisted reproduction. Although several molecular markers of oocyte quality are known, translating these into cellular functions remains challenging, largely due to the poor correlation between mRNA level and protein synthesis. Unlike most somatic cells, the oocyte can store mRNA for days, with transcriptional activity remaining at a halt during the 4-5 days beginning before ovulation and ending with embryonic genome activation. This review provides an overview of the transcriptomic data obtained from oocytes of different quality as well as interesting avenues to explore in order to improve our understanding of oocyte competence. PMID:24233546

  14. Developmental competence of equine oocytes: impacts of zona pellucida birefringence and maternally derived transcript expression.

    PubMed

    Mohammadi-Sangcheshmeh, Abdollah; Held, Eva; Rings, Franca; Ghanem, Nasser; Salilew-Wondim, Dessie; Tesfaye, Dawit; Sieme, Harald; Schellander, Karl; Hoelker, Michael

    2014-03-01

    In the present study, equine oocytes were classified into groups of presumably high and low developmental competence according to cumulus morphology, as well as oocyte ability to metabolise brilliant cresyl blue (BCB) stain. All oocytes were evaluated individually in terms of morphometry, zona pellucida birefringence (ZPB) and relative abundance of selected candidate genes. Oocytes with an expanded cumulus (Ex), representing those with presumably high developmental competence, had a significantly thicker zona (18.2 vs 17.3m) and a significantly higher ZPB (64.6 vs 62.1) than oocytes with a compacted cumulus (Cp). Concurrently, oocytes classified as highly developmentally competent (BCB+) had a significantly thicker zona (18.8 vs 16.1m) and significantly higher ZPB (63.1 vs 61.3) compared with oocytes classified as having low developmental competence. Expression of TFAM, STAT3 and CKS2 was significantly higher in Ex compared with Cp oocytes, whereas expression of COX1, ATPV6E and DNMT1 was lower. Together, the data reveal that developmentally competent equine oocytes are larger in size, have higher ZPB values and exhibit a typical genetic signature of maternally derived transcripts compared with oocytes with lower in vitro developmental competence. PMID:23622680

  15. 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

  16. NLRP9B protein is dispensable for oocyte maturation and early embryonic development in the mouse.

    PubMed

    Peng, Hui; Lin, Xiujiao; Liu, Fang; Wang, Cheng; Zhang, Wenchang

    2015-12-18

    Nlrp9a, Nlrp9b and Nlrp9c are preferentially expressed in oocytes and early embryos in the mouse. Simultaneous genetic ablation of Nlrp9a and Nlrp9c does not affect early embryonic development, but the function of Nlrp9b in the process of oocyte maturation and embryonic development has not been elucidated. Here we show that both Nlrp9b mRNA and its protein are expressed in ovaries and the small intestine. Moreover, the NLRP9B protein was restricted to oocytes in the ovary and declined with oocyte aging. After ovulation and fertilization, NLRP9B protein was found in preimplantation embryos. Confocal microscopy demonstrated that it was mainly localized in the cytoplasm in the oocytes and blastomeres. Thus, this protein might play a role in oocyte maturation and early embryonic development. However, knockdown of Nlrp9b expression in GV-stage oocytes using RNA interference did not affect oocyte maturation or subsequent parthenogenetic development after Nlrp9b-deficient oocytes were activated. Furthermore, Nlrp9b knockdown zygotes could reach the blastocyst stage after being cultured for 3.5 days in vitro. These results provide the first evidence that the NLRP9B protein is dispensable for oocyte maturation and early embryonic development in the mouse. PMID:26411641

  17. NLRP9B protein is dispensable for oocyte maturation and early embryonic development in the mouse

    PubMed Central

    PENG, Hui; LIN, Xiujiao; LIU, Fang; WANG, Cheng; ZHANG, Wenchang

    2015-01-01

    Nlrp9a, Nlrp9b and Nlrp9c are preferentially expressed in oocytes and early embryos in the mouse. Simultaneous genetic ablation of Nlrp9a and Nlrp9c does not affect early embryonic development, but the function of Nlrp9b in the process of oocyte maturation and embryonic development has not been elucidated. Here we show that both Nlrp9b mRNA and its protein are expressed in ovaries and the small intestine. Moreover, the NLRP9B protein was restricted to oocytes in the ovary and declined with oocyte aging. After ovulation and fertilization, NLRP9B protein was found in preimplantation embryos. Confocal microscopy demonstrated that it was mainly localized in the cytoplasm in the oocytes and blastomeres. Thus, this protein might play a role in oocyte maturation and early embryonic development. However, knockdown of Nlrp9b expression in GV-stage oocytes using RNA interference did not affect oocyte maturation or subsequent parthenogenetic development after Nlrp9b-deficient oocytes were activated. Furthermore, Nlrp9b knockdown zygotes could reach the blastocyst stage after being cultured for 3.5 days in vitro. These results provide the first evidence that the NLRP9B protein is dispensable for oocyte maturation and early embryonic development in the mouse. PMID:26411641

  18. Environmental and Genetic Modifiers of squint Penetrance during Zebrafish Embryogenesis

    PubMed Central

    Pei, Wuhong; Williams, P. Huw; Clark, Matthew D.; Stemple, Derek L.; Feldman, Benjamin

    2007-01-01

    The Nodal-related subgroup of the TGFβ superfamily of secreted cytokines regulates the specification of the mesodermal and endodermal germ layers during gastrulation. Two Nodal-related proteins - Squint (Sqt) and Cyclops (Cyc) - are expressed during germ-layer specification in zebrafish. Genetic sqt mutant phenotypes have defined a variable requirement for zygotic Sqt, but not for maternal Sqt, in midline mesendoderm development. However a comparison of phenotypes arising from oocytes or zygotes injected with Sqt antisense morpholinos has suggested a novel requirement for maternal Sqt in dorsal specification. In this study we examined maternal-zygotic mutants for each of two sqt alleles and we also compared phenotypes of closely related zygotic and maternal-zygotic sqt mutants. Each of these approaches indicated there is no general requirement for maternal Sqt. To better understand the dispensability of maternal and zygotic Sqt, we sought out developmental contexts that more rigorously demand intact Sqt signalling. We found that sqt penetrance is influenced by genetic modifiers, by environmental temperature, by levels of residual Activin-like activity and by Heat-Shock Protein 90 (HSP90) activity. Therefore, Sqt may confer an evolutionary advantage by protecting early-stage embryos against detrimental interacting alleles and environmental challenges. PMID:17583692

  19. Oocyte development and fecundity type of the skipjack, Katsuwonus pelamis, in the Western Indian Ocean

    NASA Astrophysics Data System (ADS)

    Grande, Maitane; Murua, Hilario; Zudaire, Iker; Korta, Maria

    2012-10-01

    The study aims to define the oogenesis pattern of the skipjack (Katsuwonus pelamis) of the Western Indian Ocean basin in terms of oocyte growth and recruitment style. The main objective is to define the type of fecundity regulation (i.e. determinate or indeterminate) based on four lines of evidence: (a) oocyte size-frequency distribution; (b) seasonal variation of the relative number and percentage of oocyte stages, (c) diameter of the advanced vitellogenic oocytes in females in the spawning capable phase; and (d) incidence of atresia throughout the spawning season. The samples were collected from 2009 to 2010 in the Western Indian Ocean, and 673 ovaries were classified in the different reproductive phases using histological staging. Moreover, the oocyte size distribution of 93 mature individuals was described by the newly implemented image analysis method. This species showed a broad oocyte size frequency distribution with no gap formation between the primary and secondary oocyte growth stages. There was no seasonal variation in the percentage of oocyte stages in ovaries in the spawning capable phase, and the diameter of those oocytes at the most advanced vitellogenic stage was approximately constant during the sampling period. These facts provide evidence of continuous oocyte recruitment into the standing stock of developing oocytes. Moreover, when reaching the end of the active reproductive period (i.e. February and March) the prevalence of atresia increased. This is a mechanism adopted by fishes of the indeterminate fecundity type to reabsorb the surplus oocyte production. Based on the findings, we state that the skipjack in the Western Indian Ocean shows asynchronous oocyte growth and an indeterminate fecundity type.

  20. 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

  1. Birth of a domestic cat kitten produced by vitrification of lipid polarized in vitro matured oocytes.

    PubMed

    Galiguis, Jason; Gmez, 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

  2. Generation of live offspring from vitrified mouse oocytes of C57BL/6J strain.

    PubMed

    Kohaya, Natsuki; Fujiwara, Katsuyoshi; Ito, Junya; Kashiwazaki, Naomi

    2013-01-01

    In mammals, unfertilized oocytes are one of the most available stages for cryopreservation because the cryopreserved oocytes can be used for assisted reproductive technologies, including in vitro fertilization (IVF) and intracytoplasmic sperm injection. However, it has generally been reported that the fertility and developmental ability of the oocytes are reduced by cryopreservation. C57BL/6J mice, an inbred strain, are used extensively for the production of transgenic and knockout mice. If the oocytes from C57BL/6J mice can be successfully cryopreserved, the cryopreservation protocol used will contribute to the high-speed production of not only gene-modified mice but also hybrid mice. Very recently, we succeeded in the vitrification of mouse oocytes derived from ICR (outbred) mice. However, our protocol can be applied to the vitrification of oocytes from an inbred strain. The aim of the present study was to establish the vitrification of oocytes from C57BL/6J mice. First, the effect of cumulus cells on the ability of C57BL/6J mouse oocytes to fertilize and develop in vitro was examined. The fertility and developmental ability of oocyte-removed cumulus cells (i.e., denuded oocytes, or DOs) after IVF were reduced compared to cumulus oocyte complexes (COCs) in both fresh and cryopreserved groups. Vitrified COCs showed significantly (P<0.05) higher fertility and ability to develop into the 2-cell and blastocyst stages compared to the vitrified DOs with cumulus cells and vitrified DOs alone. The vitrified COCs developed to term at a high success rate, equivalent to the rate obtained with IVF using fresh COCs. Taken together, our results demonstrate that we succeeded for the first time in the vitrification of mouse oocytes from C57BL/6J mice. Our findings will also contribute to the improvement of oocyte vitrification not only in animals but also in clinical applications for human infertility. PMID:23516430

  3. Activation regimens for full-term development of rabbit oocytes injected with round spermatids.

    PubMed

    Hirabayashi, Masumi; Kato, Megumi; Kitada, Kensaku; Ohnami, Naoko; Hirao, Masao; Hochi, Shinichi

    2009-06-01

    The present study was designed to investigate the effect of activation regimens on full-term development of rabbit oocytes after round spermatid injection (ROSI). In the first series, rabbit oocytes were treated with 5 microM ionomycin before ROSI, after ROSI, or before and after ROSI. In addition, non-treated oocytes were subjected to intracytoplasmic sperm injection (ICSI) using ejaculated spermatozoa. Cleavage rate of ROSI oocytes activated before and after ROSI (55%) was comparable with that of ICSI oocytes (60%), and significantly higher than those of ROSI oocytes activated either before or after ROSI (29-39%; P < 0.05). No offspring were produced by transfer of the cleaving ROSI oocytes, while 8% of the cleaving ICSI oocytes transferred gave birth to offspring. In the second series, oocytes were exposed to 5, 10, or 20 microM ionomycin, followed by ROSI, 5 microM ionomycin treatment, and incubation with 5 microg/ml cycloheximide (CHX) + 2 mM 6-dimethylaminopurine (DMAP). Significantly higher cleavage rates were derived from oocytes activated with 10 and 20 microM ionomycin before ROSI (91% and 82%, respectively; P < 0.05) compared to those activated with 5 microM ionomycin before ROSI (53%). Live offspring were obtained when the cleaving ROSI oocytes with the initial ionomycin treatment at 5 and 10 microM were transferred (offspring rate 2% and 4%, respectively). These activation regimens, however, were not valid for the ROSI using cryopreserved round spermatids. In conclusion, rabbit ROSI oocytes were capable of developing into full-term when the oocytes were activated with a combined treatment of ionomycin and CHX/DMAP. PMID:19062169

  4. Generation of Live Offspring from Vitrified Mouse Oocytes of C57BL/6J Strain

    PubMed Central

    Kohaya, Natsuki; Fujiwara, Katsuyoshi; Ito, Junya; Kashiwazaki, Naomi

    2013-01-01

    In mammals, unfertilized oocytes are one of the most available stages for cryopreservation because the cryopreserved oocytes can be used for assisted reproductive technologies, including in vitro fertilization (IVF) and intracytoplasmic sperm injection. However, it has generally been reported that the fertility and developmental ability of the oocytes are reduced by cryopreservation. C57BL/6J mice, an inbred strain, are used extensively for the production of transgenic and knockout mice. If the oocytes from C57BL/6J mice can be successfully cryopreserved, the cryopreservation protocol used will contribute to the high-speed production of not only gene-modified mice but also hybrid mice. Very recently, we succeeded in the vitrification of mouse oocytes derived from ICR (outbred) mice. However, our protocol can be applied to the vitrification of oocytes from an inbred strain. The aim of the present study was to establish the vitrification of oocytes from C57BL/6J mice. First, the effect of cumulus cells on the ability of C57BL/6J mouse oocytes to fertilize and develop in vitro was examined. The fertility and developmental ability of oocyte-removed cumulus cells (i.e., denuded oocytes, or DOs) after IVF were reduced compared to cumulus oocyte complexes (COCs) in both fresh and cryopreserved groups. Vitrified COCs showed significantly (P<0.05) higher fertility and ability to develop into the 2-cell and blastocyst stages compared to the vitrified DOs with cumulus cells and vitrified DOs alone. The vitrified COCs developed to term at a high success rate, equivalent to the rate obtained with IVF using fresh COCs. Taken together, our results demonstrate that we succeeded for the first time in the vitrification of mouse oocytes from C57BL/6J mice. Our findings will also contribute to the improvement of oocyte vitrification not only in animals but also in clinical applications for human infertility. PMID:23516430

  5. Developmental competence of immature oocytes aspirated from antral follicles in patients with gynecological diseases

    PubMed Central

    Safian, Fereshteh; Khalili, Mohammad Ali; Karimi-Zarchi, Mojgan; Mohsenzadeh, Mehdi; Ashourzadeh, Sareh; Omidi, Marjan

    2015-01-01

    Background: In vitro maturation (IVM) of immature oocytes collected from ovary has been proposed for fertility preservation. In addition, quality of oocytes post IVM is one of the factors determining its developmental competence. By using the non-invasive Polscope system, both meiotic spindle (MS) and zona pellucida (ZP) can be assessed in living oocytes. Objective: The aim was to investigate the developmental potential of immature oocytes retrieved from ovarian tissue after IVM, as a method for fertility preservation, in patients with gynecological diseases. Materials and Methods: The ovarian cortex from 26 patients with malignant and benign diseases (21-45 years old), were obtained directly from collaborating hospitals, and transported to the IVF center on ice. In total 61 immature oocytes were aspirated, of which 18 (29.5%) were degenerated and discarded. The remaining 43 (70.5%) healthy oocytes were cultured in IVM culture media for 48 hr. The rate of maturity was assessed, and the ZP birefringence and MS were imaged with Polscope technology. Results: Overall 43 immature oocytes underwent IVM technology, of which 30.2% reached viable metaphase II (MII) oocytes. The ovarian tissues of 9 (34.6%) women were lacking oocytes at any stage. During polarized light microscopy examination, MS could be visualized only in one of the MII oocytes, but high ZP birefringence’s were observed in the majority of the oocytes post IVM (61.5%). Conclusion: Oocytes maturation post IVM from unstimulated ovaries showed a good developmental competence in gynecologic patients. Further studies should be performed to advance the oocyte maturation program, such as co-culture system, for fertility preservation. PMID:26568754

  6. G-protein coupled estrogen receptor (GPER) inhibits final oocyte maturation in common carp, Cyprinus carpio.

    PubMed

    Majumder, Suravi; Das, Sumana; Moulik, Sujata Roy; Mallick, Buddhadev; Pal, Puja; Mukherjee, Dilip

    2015-01-15

    GPR-30, now named as GPER (G protein-coupled estrogen receptor) was first identified as an orphan receptor and subsequently shown to be required for estrogen-mediated signaling in certain cancer cells. Later studies demonstrated that GPER has the characteristics of a high affinity estrogen membrane receptor on Atlantic croaker and zebra fish oocytes and mediates estrogen inhibition of oocyte maturation in these two distantly related teleost. To determine the broad application of these findings to other teleost, expression of GPER mRNA and its involvement in 17?-estradiol mediated inhibition of oocyte maturation in other cyprinid, Cyprinus carpio was investigated. Carp oocytes at pre-vitellogenic, late-vitellogenic and post-vitellogenic stages of development contained GPER mRNA and its transcribed protein with a maximum at late-vitellogenic oocytes. Ovarian follicular cells did not express GPER mRNA. Carp oocytes GPER mRNA was essentially identical to that found in other perciformes and cyprinid fish oocytes. Both spontaneous and 17,20?-dihydroxy-4-pregnen-3-one (17,20?-P)-induced oocyte maturation in carp was significantly decreased when they were incubated with either E2, or GPER agonist G-1. On the other hand spontaneous oocyte maturation was significantly increased when carp ovarian follicles were incubated with an aromatase inhibitor, fadrozole, GPER antagonist, G-15 and enzymatic removal of the ovarian follicle cell layers. This increase in oocyte maturation was partially reversed by co-treatment with E2. Consistent with previous findings with human and fish GPR30, E2 treatment in carp oocytes caused increase in cAMP production and simultaneously decrease in oocyte maturation, which was inhibited by the addition of 17,20?-P. The results suggest that E2 and GPER play a critical role in regulating re-entry in to meiotic cell cycle in carp oocytes. PMID:25485460

  7. 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.

  8. Diaphragmatic herniation after penetrating trauma.

    PubMed

    Degiannis, E; Levy, R D; Sofianos, C; Potokar, T; Florizoone, M G; Saadia, R

    1996-01-01

    A study was made of 45 patients with diaphragmatic herniation after penetrating trauma. In 29 the diagnosis was established during the first admission (early presentation) and in 16 during a subsequent admission (delayed presentation). The mortality rate in the early presentation group was 3 per cent compared with 25 per cent in the delayed presentation group. The presence of gangrenous or perforated abdominal viscus in the chest cavity was the single most common and severe aggravating factor. The need for diagnosis of diaphragmatic herniation during the initial admission is emphasized. As isolated diaphragmatic injuries provide few helpful clinical features to aid diagnosis, appropriate investigations and good follow-up are of paramount importance in preventing late herniation of intra-abdominal viscera through a penetrating diaphragmatic injury. PMID:8653376

  9. Developmental potential of pig embryos reconstructed by use of sow versus pre-pubertal gilt oocytes after somatic cell nuclear transfer.

    PubMed

    Li, Juan; Pedersen, Hanne Skovsgaard; Li, Rong; Adamsen, Janne; Liu, Ying; Schmidt, Mette; Purup, Stig; Callesen, Henrik

    2014-08-01

    In this study, the developmental ability of cloned embryos using gilt versus sow oocytes was evaluated under the hypothesis that the efficiency of nuclear transfer using gilt oocytes was lower than that of sow oocytes, but that it could be optimized. Five experiments were performed with routine production of cloned embryos with sow oocytes serving as the control. Results showed that: Experiment 1: Blastocyst rates of cloned embryos with gilt oocytes was about half compared with control. Experiment 2: An extended maturation time of 48 h used for gilt oocytes resulted in lower blastocyst rates after cloning. Experiment 3: Development of cloned embryos with gilt oocytes was improved by co-culture with sow oocytes. Experiment 4: After maturation of gilt oocytes using follicular fluid from gilt instead of sow, the oocytes were sorted into large and small oocytes, and after cloning, blastocyst rates were higher using large gilt oocytes compared with small oocytes; however, the rate remained lower compared with control. Experiment 5: Six sow recipients received a total of 503 morulae and blastocysts cloned from gilt oocytes (four recipients) and 190 cloned from sow oocytes (two recipients). All recipients became pregnant and went to term, resulting in 26 (gilt oocytes) and six (sow oocytes) piglets. In conclusion, results confirmed that nuclear transfer efficiency was higher using sow versus gilt oocytes, but the use of gilt oocytes can be optimized by sorting after ooplasm size following maturation and by maturing gilt and sow oocytes together. PMID:23331714

  10. 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…

  11. 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

  12. Penetrating eye injury in war.

    PubMed

    Biehl, J W; Valdez, J; Hemady, R K; Steidl, S M; Bourke, D L

    1999-11-01

    The percentage of penetrating eye injuries in war has increased significantly in this century compared with the total number of combat injuries. With the increasing use of fragmentation weapons and possibly laser weapons on the battle-field in the future, the rate of eye injuries may exceed the 13% of the total military injuries found in Operations Desert Storm/Shield. During the Iran-Iraq War (1980-1988), eye injuries revealed that retained foreign bodies and posterior segment injuries have an improved prognosis in future military ophthalmic surgery as a result of modern diagnostic and treatment modalities. Compared with the increasing penetrating eye injuries on the battlefield, advances in ophthalmic surgery are insignificant. Eye armor, such as visors that flip up and down and protect the eyes from laser injury, needs to be developed. Similar eye protection is being developed in civilian sportswear. Penetrating eye injury in the civilian sector is becoming much closer to the military model and is now comparable for several reasons. PMID:10578588

  13. 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

  14. Autoradiographic Assessment of Blood Flow Heterogeneity in the Hamster Heart

    PubMed Central

    Stapleton, Dwight D.; Moffett, Tyler C.; Baskin, Denis G.; Bassingthwaighte, James B.

    2010-01-01

    Objective Provide regional flow measurement in the hearts of small mammals using a new, higher-resolution technique based on the deposition of a molecular marker. Methods We determined the instantaneous extraction and retention of the “molecular microsphere” radiolabeled desmethylimipramine in retrogradely perfused hamster hearts. In a separate series of experiments, autoradiography was used to measure regional myocardial deposition densities in hamster hearts of about 0.5 g with spatial area resolution of 16 × 16 μm. Results Radiolabeled desmethylimipramine is almost 100% extracted during a single transcapillary passage and is retained in the tissue for many minutes. Autoradiographic images demonstrated a spatial flow heterogeneity with standard deviations of 31 ± 4% of the mean flow (N = 5) in 16 × 16 × 20-μm3 voxels. This is equivalent to the projections made using fractal relationships from cruder observations obtained with microspheres in the hearts of baboons, sheep, and rabbits. Conclusion Autoradiography using a molecular deposition marker provides quantitative information on myocardial flow heterogeneities with resolution at the size of cardiac myocytes. Because the regions resolved are smaller than the volume of regions supplied by single arterioles, the results must slightly exaggerate the true heterogeneity of regional flows. PMID:8748951

  15. 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.

  16. 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

  17. 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.

  18. Sex differences in Siberian hamster ultradian locomotor rhythms

    PubMed Central

    Prendergast, Brian J.; Stevenson, Tyler J.; Zucker, Irving

    2014-01-01

    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

  19. Encephalomyocarditis (EMC) virus-induced orchitis in Syrian hamsters.

    PubMed

    Hirasawa, K; Takeda, M; Matsuzaki, H; Doi, K

    1991-12-01

    Testes of 8-week-old male Syrian hamsters which were inoculated intraperitoneally with 10(5) plaque-forming units of the D variant of encephalomyocarditis virus (EMC-D) were examined virologically and histologically. Viral replication was detected from 1 day post inoculation (1 DPI), became more prominent 3 DPI, and was no longer demonstrated 7 DPI. The weight of testis decreased in course of time and it was about 2% of that of control 6 weeks post inoculation (6 WPI). Histopathologically, degeneration and/or necrosis of germinal cells and spermatogonia were observed in many seminiferous tubules of all hamsters 3 DPI. At 7 DPI, luminal obstruction by cellular debris and subsequent replacement of them by mesenchymal cells were common in mildly atrophic tubules surrounded with inflammatory cells. Thereafter, atrophy of seminiferous tubules became severer with the lapse of time and, in addition to plasma cell infiltration, apparent increase in the number of Leydig cells was found in the interstices. No regenerative signs of germinal epithelia were detected by 6 WPI. This is the first report of EMC virus-induced orchitis. PMID:1662983

  20. Encephalomyocarditis (EMC) virus-induced orchitis in Syrian hamsters.

    PubMed Central

    Hirasawa, K.; Takeda, M.; Matsuzaki, H.; Doi, K.

    1991-01-01

    Testes of 8-week-old male Syrian hamsters which were inoculated intraperitoneally with 10(5) plaque-forming units of the D variant of encephalomyocarditis virus (EMC-D) were examined virologically and histologically. Viral replication was detected from 1 day post inoculation (1 DPI), became more prominent 3 DPI, and was no longer demonstrated 7 DPI. The weight of testis decreased in course of time and it was about 2% of that of control 6 weeks post inoculation (6 WPI). Histopathologically, degeneration and/or necrosis of germinal cells and spermatogonia were observed in many seminiferous tubules of all hamsters 3 DPI. At 7 DPI, luminal obstruction by cellular debris and subsequent replacement of them by mesenchymal cells were common in mildly atrophic tubules surrounded with inflammatory cells. Thereafter, atrophy of seminiferous tubules became severer with the lapse of time and, in addition to plasma cell infiltration, apparent increase in the number of Leydig cells was found in the interstices. No regenerative signs of germinal epithelia were detected by 6 WPI. This is the first report of EMC virus-induced orchitis. Images Fig. 3 Fig. 4 Fig. 5 Fig. 6 Fig. 7 Fig. 8 Fig. 9 PMID:1662983

  1. Histogenesis of exocrine pancreatic cancer in the hamster model.

    PubMed Central

    Pour, P M

    1984-01-01

    There is strong evidence that induced pancreatic adenomas and carcinomas derive from ductal and ductular cells in the pancreas. We base our beliefs on our knowledge of the embryology and histology of the pancreas in Syrian golden hamsters, along with the sequential alterations that occur during exocrine pancreatic tumor formation. This concept also has been supported by much experimental evidence, including autoradiographic, immunologic and in vitro studies. We also present other viewpoints on the origin of pancreatic cancer histogenesis and outline certain areas of disagreement. We report the development of acinar cell lesions under certain experimental dietary conditions in hamsters (the lesions resemble those commonly seen in the rat pancreatic tumor model) and the nature of these lesions. Images FIGURE 1. FIGURE 2. FIGURE 3. FIGURE 4. FIGURE 5. FIGURE 6. FIGURE 7. FIGURE 8. FIGURE 9. FIGURE 10. FIGURE 11. FIGURE 12. FIGURE 13. FIGURE 14. FIGURE 15. FIGURE 16. FIGURE 17. FIGURE 18. FIGURE 19. FIGURE 20. FIGURE 21. FIGURE 22. FIGURE 23. FIGURE 24. FIGURE 25. FIGURE 26. FIGURE 27. FIGURE 28. FIGURE 29. FIGURE 30. PMID:6236973

  2. The virulence of some strains of BCG for golden hamsters

    PubMed Central

    Bunch-Christensen, K.; Ladefoged, A.; Guld, J.

    1970-01-01

    In principle, a strain of BCG used for the preparation of live vaccine should retain a moderate residual virulence. The inoculation of golden hamsters with large doses of BCG causes progressive, fatal disease but not all strains are equally active. In a previous paper, the present authors gave data from exp