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1

Cryopreservation of hamster oocytes: effects of vitrification or freezing on human sperm penetration of zona-free hamster oocytes.  

PubMed

Three experiments were conducted for evaluation of the efficacy of conventional freezing or vitrification of hamster oocytes for use in a human sperm penetration assay (hSPA). In experiment 1, oocytes were cryopreserved and evaluated for survival on the basis of morphologic criteria. Survival of vitrified oocytes and that of frozen oocytes were not different, whereas all cryopreserved groups had lower survival than noncryopreserved controls. In experiment 2, oocytes were conventionally frozen or vitrified and evaluated in an hSPA. Vitrified oocytes had a lower frequency of sperm penetration than frozen oocytes, and all cryopreserved groups had lower penetration rates than untreated controls. In experiment 3, oocytes were exposed to the cryoprotectant used to vitrify (VS1) or freeze (DMSO) but not cooled prior to evaluation in an hSPA. Exposure to DMSO but not VS1 reduced hSPA values. It is concluded from these experiments that while all cryopreserved oocytes do not survive, at current stages of development conventionally frozen oocytes perform better than vitrified oocytes in the hSPA and losses associated with conventional freezing procedures may be related to cryoprotectant exposure, whereas vitrification losses are more probably due to events associated with rapid cooling and/or warming of the oocytes. PMID:3732535

Critser, J K; Arneson, B W; Aaker, D V; Ball, G D

1986-08-01

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

3

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...

4

Age-Associated Metabolic and Morphologic Changes in Mitochondria of Individual Mouse and Hamster Oocytes  

PubMed Central

Background In human oocytes, as in other mammalian ova, there is a significant variation in the pregnancy potential, with approximately 20% of oocyte-sperm meetings resulting in pregnancies. This frequency of successful fertilization decreases as the oocytes age. This low proportion of fruitful couplings appears to be influenced by changes in mitochondrial structure and function. In this study, we have examined mitochondrial biogenesis in both hamster (Mesocricetus auratus) and mouse (Mus musculus) ova as models for understanding the effects of aging on mitochondrial structure and energy production within the mammalian oocyte. Methodology/Principal Findings Individual metaphase II oocytes from a total of 25 young and old mice and hamsters were collected from ovarian follicles after hormone stimulation and prepared for biochemical or structural analysis. Adenosine triphosphate levels and mitochondrial DNA number were determined within individual oocytes from young and old animals. In aged hamsters, oocyte adenosine triphosphate levels and mitochondrial DNA molecules were reduced 35.4% and 51.8%, respectively. Reductions of 38.4% and 44% in adenosine triphosphate and mitochondrial genomes, respectively, were also seen in aged mouse oocytes. Transmission electron microscopic (TEM) analysis showed that aged rodent oocytes had significant alterations in mitochondrial and cytoplasmic lamellae structure. Conclusions/Significance In both mice and hamsters, decreased adenosine triphosphate in aged oocytes is correlated with a similar decrease in mtDNA molecules and number of mitochondria. Mitochondria in mice and hamsters undergo significant morphological change with aging including mitochondrial vacuolization, cristae alterations, and changes in cytoplasmic lamellae.

Simsek-Duran, Fatma; Li, Fang; Ford, Wentia; Swanson, R. James; Jones, Howard W.; Castora, Frank J.

2013-01-01

5

Characterization of an oviductal glycoprotein associated with the ovulated hamster oocyte  

Microsoft Academic Search

Hamster oviducts in culture incorporate (³⁵S)-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

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

1988-01-01

6

Human sperm chromosome analysis after subzonal sperm insemination of hamster oocytes  

SciTech Connect

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.

Cozzi, J. [Medical School of Grenoble (France)

1994-09-01

7

Parthenogenetic activation of pig oocytes with calcium ionophore and the block to sperm penetration after activation.  

PubMed

Calcium ionophore A23187 can parthenogenetically activate oocytes in many animals. The present study was designed to analyze functionally the mechanism of A23187 activation of pig oocytes matured in vitro. In experiment 1, effects of the concentration of A23187 on intracellular calcium transients, cortical granule (CG) exocytosis, nuclear activation, and zona reaction, which was determined by zona hardening and sperm penetrability, were examined. Cumulus-free oocytes were exposed to 0-100 microM A23187 for 5 min. It was found that the amplitude of the intracellular calcium transients, percentage of CG exocytosis, and percentage of pronuclear formation were increased in a concentration-dependent manner. The time for dissolution of zona pellucida (ZP) was increased in the oocytes treated with 25-100 microM A23187. Penetration of ZP-intact oocytes by spermatozoa was decreased and only 3-4% of oocytes were penetrated by spermatozoa after 50-100 microM A23187 treatment. In experiment 2, oocytes were treated with 100 microM A23187 for 5 min and then cultured for 10 min or 3.5 h before insemination. No difference in penetration rates was observed between the two groups of oocytes (12.0% vs. 12.2%), but the penetration rates were significantly lower than those in controls (85.2% vs. 82.4%). In experiment 3, treatment of oocytes with 100 microM A23187 for 5 min was followed by removal of the ZP from a portion of the oocytes. ZP-intact and ZP-free oocytes were then inseminated for examination of sperm penetration. One of 65 (2%) oocytes with ZP and 48 of 52 (92%) oocytes without ZP were penetrated by spermatozoa. These results indicate that activation of pig oocytes by A23187 is the result of A23187-induced intracellular calcium increase and that A23187-induced cortical reaction can prevent sperm penetration of ZP-intact oocytes, but not ZP-free oocytes. PMID:9623593

Wang, W H; Machty, Z; Abeydeera, L R; Prather, R S; Day, B N

1998-06-01

8

The rates of premature chromosome condensation and embryo development after injection of irradiated sperms into hamster oocytes.  

PubMed

Background: Irradiation is one of the major causes of induced sperm DNA damage. Various studies suggested a relation between sperm DNA damage and fertilization rate after intra-cytoplasmic sperm injection (ICSI). Objective: In this study, fertilization rate and premature chromosome condensation (PCC) formation after ICSI of hamster oocytes with irradiated sperms from normal and oligosperm individuals was investigated. Materials and Methods: Human sperms were classified according to counts to normal and oligosperm. Ten samples were used for each group. Golden hamster oocytes were retrieved after super ovulation by PMSG and HCG injection. From retrieved oocytes, 468 were in metaphase II. Control and 4 Gy gamma irradiated sperms were then injected into oocytes. After pronuclei formation in injected oocytes and formation of 8 cells embryos, slides were prepared using Tarkowskie's standard air-drying technique. The frequency of embryos and PCC were analyzed using 1000 microscope after staining in 5% Giemsa. Results: The extent of embryo development in oocytes injected by irradiated sperms was lower than those injected by non-irradiated sperms (p=0.0001). The frequency of PCC in failed fertilized oocytes was significantly higher in oligosperms (46%) compared with normal ones (0%), but there was no significant difference between irradiated and non-irradiated samples in each group (p=0.12). Conclusion: The results showed that irradiation of sperms might influence the fertilization outcome possibly due to sperm DNA damage. One possible cause of precluding oocytes from fertilization in oligosperm individuals might be the formation of PCC. PMID:24639771

Moghbelinejad, Sahar; Mozdarani, Hossein; Rezaeian, Zahra

2013-05-01

9

The rates of premature chromosome condensation and embryo development after injection of irradiated sperms into hamster oocytes  

PubMed Central

Background: Irradiation is one of the major causes of induced sperm DNA damage. Various studies suggested a relation between sperm DNA damage and fertilization rate after intra-cytoplasmic sperm injection (ICSI). Objective: In this study, fertilization rate and premature chromosome condensation (PCC) formation after ICSI of hamster oocytes with irradiated sperms from normal and oligosperm individuals was investigated. Materials and Methods: Human sperms were classified according to counts to normal and oligosperm. Ten samples were used for each group. Golden hamster oocytes were retrieved after super ovulation by PMSG and HCG injection. From retrieved oocytes, 468 were in metaphase II. Control and 4 Gy gamma irradiated sperms were then injected into oocytes. After pronuclei formation in injected oocytes and formation of 8 cells embryos, slides were prepared using Tarkowskie's standard air-drying technique. The frequency of embryos and PCC were analyzed using 1000 microscope after staining in 5% Giemsa. Results: The extent of embryo development in oocytes injected by irradiated sperms was lower than those injected by non-irradiated sperms (p=0.0001). The frequency of PCC in failed fertilized oocytes was significantly higher in oligosperms (46%) compared with normal ones (0%), but there was no significant difference between irradiated and non-irradiated samples in each group (p=0.12). Conclusion: The results showed that irradiation of sperms might influence the fertilization outcome possibly due to sperm DNA damage. One possible cause of precluding oocytes from fertilization in oligosperm individuals might be the formation of PCC.

Moghbelinejad, Sahar; Mozdarani, Hossein; Rezaeian, Zahra

2013-01-01

10

Characterization of an oviductal glycoprotein associated with the ovulated hamster oocyte  

SciTech Connect

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.

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

1988-04-01

11

OOCYTE MATURATION: ABERRANT POST FUSION RESPONSES OF THE RABBIT PRIMARY OOCYTE TO PENETRATING SPERMATOZOA  

Microsoft Academic Search

SUMMARY Primary oocytes cannot be fertilized normally; they begin to develop this capacity as meiosis resumes. To elucidate the changes involved in acquisition of their fertilizability, rabbit primary oocytes displaying a germinal vesicle (GV oocytes) were placed in Fallopian tubes inseminated previously with spermatozoa, recovered 2-5 h later and examined by light and electron micro- scopy. At least 4 aspects

M. BERRIOS; J. M. BEDFORDf

12

Fetal calf serum increased the zona pellucida penetrability of rat oocytes matured in vitro.  

PubMed

We examined the effect of fetal calf serum (FCS) on meiotic division, subsequent fertilization, and first cleavage to the 2-cell stage of rat oocytes during in vitro maturation. FCS had no effect on the nuclear progression from dictiate to metaphase of the second maturation in vitro and, FCS had no effect on the first cleavage to the 2-cell stage of fertilized oocytes. However, FCS efficiently increased penetration rate of oocytes and shortened the time required for dissolution of the zona pellucida by alpha-chymotrypsin. These results showed that FCS did not affect cytoplasmic maturation necessary for oocytes to develop to the 2-cell stages. We found that FCS only affects the zona pellucida and does not affect the nucleus or cytoplasm of rat oocytes. FCS may prevent hardening of the zona pellucida. PMID:2244475

Fujii, Y; Yoshioka, T; Sasaki, J

1990-08-01

13

Structure of the cumulus matrix and zona pellucida in the golden hamster: A new view of sperm interaction with oocyte-associated extracellular matrices  

Microsoft Academic Search

Hamster oocyte-cumulus complexes (OCC), with and without sperm, were structurally analyzed by light- and electron microscopy using freeze substitution. This method has yielded a clear picture of the extracellular oocyte investments, the cumulus cell matrix and the zona pellucida. The cumulus matrix has an overall homogeneous fibrillar structure which appears to attach to cumulus cells at their filopodial extensions. The

Ashley I. Yudin; Gary N. Cherr; David F. Katz

1988-01-01

14

Bovine oocyte quality in relation to ultrastructural characteristics of zona pellucida, polyspermic penetration and developmental competence.  

PubMed

In the present study, the effect of bovine oocyte quality related to ultrastructural characteristics of zona pellucida (ZP), polyspermic penetration and embryo developmental competence was evaluated. Cumulus-oocyte complexes were punctioned from 453 ovaries, classified as 1, 2, 3 and 4 according to their morphological aspect, matured for 24 h and then divided into two groups. In group A, oocytes were fixed in 2.5% glutaraldehyde and 0.1 m sodium cacodylate and examined under a scanning electron microscope. Photomicrographs were taken and ZP's pores were evaluated in squares of 6.4-microm width. In group B, oocytes were fertilized in vitro. After 48 h, non-cleaved oocytes were fixed for polyspermy evaluation. On days 7, 9 and 10, embryos were classified as developed (blastocysts and hatched blastocysts). Results showed that quality 1 oocytes revealed a ZP pore diameter of 0.50 +/- 0.07 microm, which was smaller than the observed on oocytes of quality 2 (0.83 +/- 0.10 microm), quality 3 (1.02 +/- 0.22 microm) and quality 4 (1.38 +/- 0.59 microm) (p < or = 0.05). For In Vitro Fertilization (IVF), results showed that embryos originating from oocytes classed as 3 and 4 had lower cleavage rate (68.4% and 43.8%) than those belonging to class 1 and 2 (79.5% and 69.3%) (p < or = 0.05). None oocyte classified as 3 and 4 developed to hatch blastocysts, while for oocytes belonging to quality 1 and 2, these values were, respectively, 15.2% and 12.5%. Concerning polyspermy, oocytes class 1 and 2 had lower polyspermic penetration than those belonging to class 3 and 4 (respectively 4.1%, 4.5%, 11.1% and 9.8%, for class 1, 2, 3 and 4). In conclusion, the present study demonstrated that oocytes with low qualities result in lower developmental competence and with high percentage of polyspermy after IVF, which can be the result of the ZP structure such as the number and the pore's diameter. PMID:18484959

Santos, P; Chaveiro, A; Simes, N; Moreira da Silva, F

2008-12-01

15

Ability of Catalonian donkey sperm to penetrate zona pellucida-free bovine oocytes matured in vitro.  

PubMed

An experiment was designed to study the interaction between fresh/frozen-thawed donkey spermatozoa and zona pellucida (ZP)-free bovine oocytes in an attempt to develop a model for assessing cryopreserved Catalonian donkey sperm function. Semen from five donkeys was collected using an artificial vagina. Sperm motility and viability were immediately assessed and the semen sample cryopreserved. Sperm viability and motility were then reassessed immediately after thawing. The motion characteristics of the fresh and frozen-thawed spermatozoa were determined using a computer-assisted sperm analysis system. In vitro-matured cow oocytes were inseminated with different percent live donkey sperm (high (>60%) or low (<40%) viability donkey sperm). After 18h of co-incubation, the oocytes were fixed, stained with 4',6-diamidino-2-phenylindole (DAPI) and examined for sperm penetration, the number of penetrated spermatozoa per oocyte, and male pronucleus formation. Frozen-thawed spermatozoa from high viability semen showed significantly lower VCL, VAP and mean ALH values than did high viability fresh spermatozoa. In contrast, frozen-thawed spermatozoa of low viability had significantly higher velocity values than fresh spermatozoa of low viability. A significant positive correlation (P<0.01) was detected between percentage fertilization and viability (r=0.84), and between percentage fertilization and certain CASA parameters (VAP, r=0.56; VCL, r=0.61 and mean ALH, r=0.68). Fresh or frozen-thawed high viability spermatozoa penetrated 90.1% and 85.4% of bovine oocytes respectively. Lower rates of penetration were observed for fresh and frozen-thawed low viability spermatozoa (34% and 22.5% respectively). The donkey spermatozoa were able to fuse with the oolema and even to decondense and form the male pronucleus (85-94%). Larger numbers of penetrated spermatozoa per oocyte were recorded when high viability sperm samples were used, whether fresh (3.02 vs. 1.12 for low viability sperm) or frozen-thawed (3.41 vs. 1.47). Consequently, low viability sperm samples showed higher percentages of monospermic penetration (91.17% and 61.97% for fresh and frozen-thawed sperm samples respectively). These findings suggest that bovine oocytes provide a useful model for assessing the penetration potential of frozen-thawed donkey sperm. PMID:19748750

Taberner, E; Morat, R; Mogas, T; Mir, J

2010-04-01

16

Sperm penetration to the zona pellucida of an oocyte: a computational model incorporating acrosome reaction.  

PubMed

For fertilisation to occur, a spermatozoon needs to cross the zona pellucida (ZP), which is a glycoprotein layer surrounding the oocyte. Crossing the ZP requires an acrosome reaction (AR) where enzymes released from the spermatozoon head locally digest and soften the ZP so that the spermatozoon can penetrate deeper. Here, a biomechanical sperm-oocyte interaction model that considers the AR using the finite element method was formulated. This modelling is used to determine which of the following factors directly contribute to the crossing of the ZP: local ZP softening by AR, sperm head shape, ZP hardening elsewhere than in the AR site, ZP thickness and sperm hyperactivation (more flagellar beating). It has been found that an AR softening the ZP to over one-tenth of its basal stiffness is important for successful sperm penetration, and that 'sharper' heads have a biomechanical advantage in penetrating deeper. The approach is promising for understanding this exciting stage of reproduction. PMID:23477851

Kozlovsky, Pavel; Gefen, Amit

2013-10-01

17

Effects of White Blood Cells on the In Vitro Penetration of Zona-free Hamster Eggs by Human Spermatozoa  

Microsoft Academic Search

The presence of white blood cells in semen has been associated with male infertility. Previous studies indi- cate that pyospermia occurs in conjunction with de- creases in sperm motility, number of normal sperm forms, and penetration rates in the zona-free hamster egg sperm penetration assay. We have evaluated the relationship of seminal white blood cells and sperm function, as reflected

DONALD K. MARUYAMA; RALPH W. HALE; B. JANE ROGERS

18

Hamsters  

NSDL National Science Digital Library

Scientists believe that most behaviors, from fighting to mating, are controlled by brain chemistry. In this Science Update, you'll hear how researchers are using hamsters to understand the roots of aggression.

Science Update;

2000-03-15

19

Platelet activating factor improves the in vitro penetration of zona free hamster eggs by buffalo (Bubalus bubalis) spermatozoa.  

PubMed

Twelve buffalo bulls of Murrah breed, selected on the basis of their conception rates, were classified into low-, moderate- and high-fertility groups. Frozen semen was thawed and treated with 200 microM platelet activating factor (PAF) for 15 min at 37 degrees C and 5% CO2. In both treated and control (no PAF) semen samples (five replicates per bull), the following were assessed: motility, acrosome reaction (AR) evaluation (for 10 replicates of each bull), and zona-free hamster oocyte penetration test--to determine aspects of fertilization in vitro, viz., sperm attached per ovum (SA/O), fertilization percent (FP), fertilization index (FI), and polyspermic ova (PO). There was an effect of group (P < 0.01) on all parameters; all except motility were increased by PAF treatment. However, the group X treatment interaction was not significant for any parameter. The overall mean values of motility, AR, SA/O, FP, FI, and PO, for controls, treated spermatozoa and (net change) were: 42.89 +/- 0.85, 36.65 +/- 0.85, (-6.24); 28.94 +/- 0.46, 61.44 +/- 0.58, (32.50); 126 +/- 2, 145 +/- 2, (19); 74.21 +/- 1.59, 89.11 +/- 1.18, (14.90); 0.79 +/- 0.02, 1.10 +/- 0.03, (0.31) and 5.22 +/- 1.22, 21.69 +/- 1.88, (16.47)%, respectively. In conclusion, PAF significantly increased the AR and other aspects of fertilization, despite a small reduction in motility. PMID:15763101

Kumar, Subodh; Sharma, Arjava

2005-04-01

20

Importance of Glutathione in the Acquisition and Maintenance of Sperm Nuclear Decondensing Activity in Maturing Hamster Oocytes.  

National Technical Information Service (NTIS)

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. Previ...

S. D. Perreault R. R. Barbee V. L. Slott

1988-01-01

21

Differing sperm ability to penetrate the oocyte in vivo and in vitro as revealed using colloidal preparations.  

PubMed

The penetration ability of boar (Sus scrofa domestica) spermatozoa exposed to viscous preparations under in vivo and in vitro fertilization conditions has been examined. Experiments involving induced ovulation in prepubertal animals and surgical insemination directly into the oviduct isthmus revealed an advantage of colloidal preparations. Based on within-animal comparisons, the incidence of penetration was 100% using both spermatozoa suspended in a viscous preparation of plant extracts and spermatozoa suspended in a control medium. However, percentages of monospermy were 22.2% in 54 oocytes inseminated with the control suspension compared with 62.5% in 48 oocytes inseminated with the colloidal preparation. An in vitro study involving 355 oocytes from slaughterhouse ovaries inseminated with in vitro-capacitated spermatozoa gave similar percentages of penetrated oocytes for both the control and colloidal suspensions. In this case, however, the percentage of monospermy was 32.7% in the control group compared with 10.6% for spermatozoa suspended in the colloidal preparation. Higher mean numbers of sperm inside the oocytes and higher numbers of sperm bound to the zona pellucida were also observed with the colloidal suspensions. In vitro motility and viability for spermatozoa in the colloidal suspensions were enhanced compared with that of the control group. Lower sperm membrane lipid disorder and reactive oxygen species generation were also observed in the viscous solution. These findings suggest that viscous fluids can enhance the ability of sperm to move, bind, and penetrate the oocyte in vitro, although this influence may be masked in vivo due to the already high viscosity in the oviductal fluid close to the time of ovulation. PMID:19744704

Coy, P; Gadea, J; Rath, D; Hunter, R H F

2009-12-01

22

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...

23

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

PubMed

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

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

24

Effect of capacitation of stallion sperm with polyvinylalcohol or bovine serum albumin on penetration of bovine zona-free or partially zona-removed equine oocytes.  

PubMed

Experiments were conducted to study effects of macromolecules on stallion sperm capacitation and fertilization as determined by penetration of bovine zona-free and equine partially zona-removed oocytes. Stallion sperm were capacitated in TYH medium (modified Krebs-Ringer bicarbonate) supplemented with either 1 mg/mL of polyvinylalcohol (PVA) or 4 mg/mL of BSA. Capacitation was induced with 8 bromoadenosine cyclic monophosphate (8BrcAMP; 0.5 mM) alone or in combination with 0.1 microM of ionomycin. Intraspecies gametes were co-incubated in TYH/PVA or TYH/BSA for 18 to 20 h. For zona-free bovine oocytes, penetration rate (35%) with the combination of 8BrcAMP and ionomycin in PVA-containing medium was higher (P < 0.05) than any treatment in BSA-containing medium (5 to 6%). A similar study was conducted using equine oocytes with partially removed zonae. Sperm capacitated and used for in vitro fertilization (IVF) in PVA-containing medium had higher penetration rates (P < 0.01) than sperm in BSA-containing medium (54 vs. 11%). The effect of equine preovulatory follicular fluid on bovine oocyte penetration was assessed. Bovine oocytes were matured in tissue culture medium-199 with 0, 20, 50, or 100% equine preovulatory follicular fluid, and 1 IU/mL of equine chorionic gonadotropin. Stallion sperm were treated with 8BrcAMP + ionomycin in PVA- or BSA-containing media. The penetration rates of bovine zona-free oocytes by stallion sperm were again higher with PVA (47%) than BSA (18%; P < 0.01). Penetration rates of oocytes matured in 100% follicular fluid were higher (P < 0.05) than for oocytes matured with 0% follicular fluid. The effects of equine follicular fluid and PVA/BSA during sperm capacitation on standard bovine IVF were examined. Culture of bovine oocytes with equine follicular fluid did not affect oocyte maturation or penetration rates after IVF. Bovine sperm capacitated with heparin in PVA-containing medium yielded lower (P < 0.05) fertilization rates than those capacitated in BSA-containing medium when incubated with both zona-intact and zona-free bovine oocytes. In summary, PVA was superior to BSA for ionophore-induced capacitation of equine sperm for penetration of zona-free bovine oocytes or partially zona-removed equine oocytes, but not for standard bovine IVF with bovine sperm. Zona-free bovine oocytes may be useful for assaying in vitro capacitation and fertilization of stallion sperm. PMID:12926790

Choi, Y H; Landim-Alvarenga, F C; Seidel, G E; Squires, E L

2003-08-01

25

Vitrification solution containing DMSO and EG can induce parthenogenetic activation of in vitro matured ovine oocytes and decrease sperm penetration.  

PubMed

This study was designed to examine the reduced incidence of normal fertilization in vitrified ovine oocytes. After in vitro maturation for 24 h, the oocytes were randomly allocated into three groups: (1) untreated (control), (2) exposed to vitrification solution (VS) without being plunged into liquid nitrogen (toxicity), or (3) vitrified by open-pulled straw method (vitrification). In experiment 1, the treated and control oocytes were matured for another 2 h, and the oocytes were then in vitro fertilized for 12 h to examine sperm penetration. The percentage of monospermy in toxicity group (29.3%) and vitrification group (28.2%) dramatically decreased compared to the control group (45.0%) (P<0.05). To find the mechanism that the VS decreased the monospermy, some treated and control oocytes were used to test the distribution of CG and the resistance of zona pellucida (ZP) to 0.1% pronase E immediately (IVM 24 h), after another 2 h of maturation (IVM 26 h), and after 12 h of in vitro fertilization (IVF 12 h) respectively. Others were used to examine female pronucleus formation after 12 h of culture in fertilization medium with the absence of sperm. The results showed that the percentage of CG completely release in the oocytes (IVM 24 and 26 h) of toxicity group (41.2% and 39.9%) and vitrification group (41.7% and 51.7%) was significantly higher than that of control group (7.1% and 18.4%) (P<0.05). The ZP digestion duration in the oocytes (IVM 26 h) of the toxicity group (435.6 s) and vitrification group (422.3 s) was longer than that of control group (381.6 s) (P<0.05). The percentage of female pronucleus formation in toxicity group (58.7%) and vitrification group (63.9%) was higher than that (8.2%) of control group (P<0.05). The data above demonstrated that the VS containing DMSO and EG could parthenogenetically activate in vitro matured ovine oocytes, resulting in ZP hardening and decreased sperm penetration. PMID:17293065

Tian, Shu-Jun; Yan, Chang-Liang; Yang, Hui-Xin; Zhou, Guang-Bin; Yang, Zhong-Qiang; Zhu, Shi-En

2007-10-01

26

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

The objective of this research was to estimate the optimal timing for fertilization to achieve proper embryonic development of in vitro-matured bovine oocytes. First, cumulus-oocyte complexes were subjected to in vitro maturation (IVM) for 14-22 hr. The timing when 50% of oocytes reached metaphase II stage was estimated to be 17.5 hr after IVM start. Next, using oocytes subjected to IVM for 12-30 hr, sperm penetration was examined after 4-18 hr of in vitro fertilization (IVF). A significant negative correlation between IVM duration and the timing when 50% of oocytes were penetrated by sperm after IVF start was observed (P<0.01). Finally, oocytes subjected to 12-30 hr of IVM were inseminated and cultured for 6 days to examine embryonic development. In the group with 22 hr of IVM, the percentages of cleaved embryos and blastocysts were the highest values in all groups. According to the regression equation describing the time from nuclear maturation to sperm penetration (x) and the percentage of blastocysts (y) (y=7.23x - 0.297x(2), P<0.01), the blastocyst rate peaked when sperm penetration occurred at 12.2 hr after achieving nuclear maturation. In conclusion, under the present IVM/IVF conditions, it was estimated that oocytes acquired their highest developmental competence at about 30 hr after IVM start, and thus, the optimal IVM duration was calculated to be about 21 hr. PMID:24430663

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

2014-06-01

27

The Sperm Penetration Assay for the Assessment of Fertilization Capacity  

PubMed Central

Summary The sperm penetration assay, or zona-free hamster oocyte penetration assay is utilized to measure the ability of sperm to undergo capacitation, fuse with the egg membrane and decondense the sperm head within the cytoplasm of the oocyte resulting in the formation of the male pronucleus. The test is scored by calculation the percentage of ova that are penetrated or the average number of sperm penetrations per ovum. It has been used to identify those couples who will have a high likelihood of success with in vitro fertilization.

Hwang, Kathleen; Lamb, Dolores J.

2013-01-01

28

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...

29

Factors required during preculture of rat oocytes soon after sperm penetration for promoting their further development in a chemically defined medium.  

PubMed

Blastocyst formation in a chemically defined medium (mR1ECM) of rat oocytes soon after sperm penetration is less frequent than in those undergoing male pronuclear formation. This inhibition is released by preculturing the oocytes for a few hours in modified Krebs-Ringer bicarbonate solution (mKRB). The present study examined the effects of phosphate (Pi), bovine serum albumin (BSA) and osmolarity during preculture of sperm penetrated rat oocytes on their development to blastocysts in mR1ECM in vitro. These are the major factors that differ between mR1ECM and mKRB. When oocytes collected at 0730-0800 h on the day following mating and freed from cumulus cells were precultured for 5 h in mKRB or Pi-free mKRB and then cultured for 127 h in mR1ECM, about 73-74% of oocytes developed to blastocysts. In both media, replacement of BSA with polyvinylalcohol (PVA) or osmolarity of 246 mOsM reduced blastocyst formation compared with media containing BSA or with osmolarity of 304 mOsM; blastocyst formation was greatly inhibited when oocytes were precultured in media with PVA and osmolarity of 246 mOsM. On the other hand, when precultured in mR1ECM or mR1ECM with osmolarity of 304 mOsM or BSA instead of PVA, fewer oocytes developed to blastocysts than those precultured in Pi-free mKRB and mR1ECM with osmolarity of 304 mOsM and BSA. These results indicate that both BSA and osmolarity, but not Pi, are essential factors during preculture of rat oocytes soon after sperm penetration for promoting their further development to blastocysts in a chemically defined medium. PMID:15514459

Yang, Xin-Zhi; Han, Myung-Sook; Niwa, Koji; Iannaccone, Philip M

2004-10-01

30

Ability of Catalonian donkey sperm to penetrate zona pellucida-free bovine oocytes matured in vitro  

Microsoft Academic Search

An experiment was designed to study the interaction between fresh\\/frozen-thawed donkey spermatozoa and zona pellucida (ZP)-free bovine oocytes in an attempt to develop a model for assessing cryopreserved Catalonian donkey sperm function. Semen from five donkeys was collected using an artificial vagina. Sperm motility and viability were immediately assessed and the semen sample cryopreserved. Sperm viability and motility were then

E. Taberner; R. Morat; T. Mogas; J. Mir

2010-01-01

31

Functional characterization of the human intestinal NaPi-IIb cotransporter in hamster fibroblasts and Xenopus oocytes  

Microsoft Academic Search

The recently cloned NaPi-IIb cotransporter is an apical membrane protein that is involved in the absorption of phosphate in the intestine. To expedite functional and structural studies, the human intestinal NaPi-IIb cotransporter was stably expressed in hamster fibroblast (PS120) cells. The hNaPi-IIb cDNA stably transfected cells exhibited a 1.8-fold higher sodium-dependent phosphate uptake than vector DNA transfected cells, and had

Hua Xu; Michael Inouye; Timothy Missey; James F Collins; Fayez K Ghishan

2002-01-01

32

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

PubMed

To study peptide absorption in chickens, an intestinal peptide transporter cDNA (cPepT1) was isolated from a chicken duodenal cDNA library. The cDNA was 2914 bp long and encoded a protein of 714 amino acid residues with an estimated molecular size of 79.3 kDa and an isoelectric point of 7.48. cPepT1 protein is similar60% identical to PepT1 from rabbits, humans, mice, rats and sheep. Sixteen dipeptides, three tripeptides and four tetrapeptides that contained the essential amino acids Met, Lys and(or) Trp were used for functional analysis of cPepT1 in Xenopus oocytes and Chinese hamster ovary cells. For most di- and tripeptides tested, the substrate affinities were in the micromolar range, indicating that cPepT1 has high affinity for these peptides. Lys-Lys and Lys-Trp-Lys were exceptions, with substrate affinities in the millimolar range. Neither free amino acids nor tetrapeptides were transported by cPepT1. Northern blot analysis using a full-length cPepT1 cDNA as the probe demonstrated that cPepT1 is expressed strongly in the duodenum, jejunum and ileum, and at lower levels in kidney and ceca. The present study demonstrated for the first time the presence and functional characteristics of a peptide transport system from an avian species. PMID:11880560

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

2002-03-01

33

Differing sperm ability to penetrate the oocyte in vivo and in vitro as revealed using colloidal preparations  

Microsoft Academic Search

The penetration ability of boar (Sus scrofa domestica) spermatozoa\\u000a exposed to viscous preparations under in vivo and in vitro\\u000a fertilization conditions has been examined. Experiments involving\\u000a induced ovulation in prepubertal animals and surgical insemination\\u000a directly into the oviduct isthmus revealed an advantage of colloidal\\u000a preparations. Based on within-animal comparisons, the incidence of\\u000a penetration was 100\\\\% using both spermatozoa suspended in

P. Coy; J. Gadea; D. Rath; R. H. F. Hunter

2009-01-01

34

In vitro development of polyspermic porcine oocytes: Relationship between early fragmentation and excessive number of penetrating spermatozoa.  

PubMed

Embryo development during in vitro culture of polyspermic porcine oocytes was investigated in the present study. After in vitro fertilization (IVF) of in vitro matured oocytes, putative zygotes were centrifuged to visualize pronuclei. Two pronuclear (2PN) and poly-pronuclear (PPN) zygotes were selected and cultured in vitro. Their development to the blastocyst stage and total cell numbers, dead cell rates and ploidy at the blastocyst stage and morphology of resultant embryos after first cleavage were compared. A cleavage rate of PPN embryos was lower than that of 2PN (61.3% and 82.2%, respectively), however, the ability of cleaved embryos to develop to the blastocyst stage did not differ between the PPN and the 2PN groups (22.4% and 32.9%, respectively). Also there was no difference in total cell numbers and rates of dead cells between PPN and 2PN blastocysts. The majority of blastocysts in 2PN group were found to be diploid. In contrast, blastocysts in PPN group showed heterogeneous status in their ploidy including polyploidy and mixoploidy, whereas a remarkable proportion (31.3%) of them was found to be diploid. After the first cleavage (at 36 h after IVF), there was no difference in the number of nuclei/embryo between the two groups, nevertheless embryos in PPN group had significantly higher numbers of blastomeres than that of embryos in 2PN group, mainly due to an increased frequency of anuclear blastomeres. The present results indicate that correction of embryo ploidy in polyspermic embryos can occur during IVC. Nevertheless the frequency of partial fragmentation in polyspermic embryos is increased. PMID:17681437

Somfai, Tams; Ozawa, Manabu; Noguchi, Junko; Kaneko, Hiroyuki; Karja, Ni Wayan Kurniani; Fahrudin, Mokhamad; Nakai, Michiko; Maedomari, Naoki; Dinnys, Andrs; Nagai, Takashi; Kikuchi, Kazuhiro

2008-08-01

35

Comparison of the fertility of cryopreserved stallion spermatozoa with sperm motion analyses, flow cytometric evaluation, and zona-free hamster oocyte penetration  

Microsoft Academic Search

Stallion spermatozoa were cryopreserved in different extenders, and the correlations between laboratory assay results and sperm fertility were determined. Spermatozoa were cryopreserved in 1) a skim milk-egg yolk medium (CO); 2) a skim milk-egg yolk-sugar medium (SMEY); 3) CO after pretreatment with phosphatidylserine+cholesterol liposomes (CO + L); or 4) cooled to 5C without cryopreservation. The per cycle embryo recovery rates

K. M. Wilhelm; J. K. Graham; E. L. Squires

1996-01-01

36

Sperm attachment and penetration competence in the human oocyte: a possible aetiology of fertilization failure involving the organization of oolemmal lipid raft microdomains influenced by the ??m of subplasmalemmal mitochondria.  

PubMed

The roles of oolemmal lipid raft microdomains enriched in the ganglioside GM1 and the tetraspanin protein CD9 were investigated as causative agents in fertilization failure in human IVF where spermatozoa progress to the oolemma but fail to attach or, if attached, to penetrate. The findings show that specific configurations of GM1 lipid raft microdomains are consistent with attachment and penetration, while microdomains composed of CD9 lipid rafts, a protein known to be critical for penetration, do not appear to have a central role in the initial stages of attachment. The relative magnitude of the potential difference across the inner membrane (??m) in mitochondria localized to a stable subplasmalemmal domain appears to influence the organization of GM1 but not CD9 lipid raft microdomains in the corresponding oolemma. The findings present a novel view of how fertilization competence may be established in the human oocyte and a means by which certain fertilization failures that occur after conventional clinical IVF can be identified and explained in the unfortunate instance of fertilization arrest at the oolemma. PMID:24157131

Van Blerkom, Jonathan; Caltrider, Kyle

2013-12-01

37

Sperm penetration assay as an indicator of bull fertility.  

PubMed

To predict the fertility of frozen-thawed bull spermatozoa, a sperm penetration assay (SPA) using zona-free hamster oocytes was optimized, and the assay results were compared with data from field fertility expressed as the non-return rate (NRR). To increase sperm penetration, the spermatozoa were pre-incubated and coincubated with oocytes in media containing various concentrations of heparin (0 to 50 ?g/ml). Coincubation with 10 ?g/ml heparin showed the highest sperm penetration (P<0.05); it is considered to be the optimized SPA method. Sperm fertility index values obtained from WSPA were significantly correlated with the historic average NRR of 46 bulls (P<0.01). To determine the normal range for SPA, we established the lower limits of the sperm fertility index and set the cut-off value at 2.55, at which point the NRR was more than 70%, using the receiver operating characteristic curve. The overall accuracy for the 46 bulls was 95.7% (44/46) for both the low and high NRR, with a sensitivity of 95.5% (21/22) and a specificity of 95.8%. This protocol would make it easier to discriminate bulls according to their sperm fertilizing ability. PMID:22522227

Park, Yoo-Jin; Mohamed, El-Sayed A; Oh, Shin-Ae; Yoon, Sung-Jae; Kwon, Woo-Sung; Kim, Heung-Ruil; Lee, Myeung-Sik; Lee, Kichoon; Pang, Myung-Geol

2012-01-01

38

Effect of capacitation of stallion sperm with polyvinylalcohol or bovine serum albumin on penetration of bovine zona-free or partially zona-removed equine oocytes1  

Microsoft Academic Search

Experiments were conducted to study effects of macromolecules on stallion sperm capacita- tion and fertilization as determined by penetration of bovine zona-free and equine partially zona-removed oo- cytes. Stallion sperm were capacitated in TYH medium (modified Krebs-Ringer bicarbonate) supplemented with either 1 mg\\/mL of polyvinylalcohol (PVA) or 4 mg\\/ mL of BSA. Capacitation was induced with 8 bromoade- nosine cyclic

Y. H. Choi; F. C. Landim-Alvarenga; G. E. Seidel Jr; E. L. Squires

2003-01-01

39

Inhibiting Sperm Pyruvate Dehydrogenase Complex and Its E3 Subunit, Dihydrolipoamide Dehydrogenase Affects Fertilization in Syrian Hamsters  

PubMed Central

Background/Aims The importance of sperm capacitation for mammalian fertilization has been confirmed in the present study via sperm metabolism. Involvement of the metabolic enzymes pyruvate dehydrogenase complex (PDHc) and its E3 subunit, dihydrolipoamide dehydrogenase (DLD) in hamster in vitro fertilization (IVF) via in vitro sperm capacitation is being proposed through regulation of sperm intracellular lactate, pH and calcium. Methodology and Principal Findings Capacitated hamster spermatozoa were allowed to fertilize hamster oocytes in vitro which were then assessed for fertilization, microscopically. PDHc/DLD was inhibited by the use of the specific DLD-inhibitor, MICA (5-methoxyindole-2-carboxylic acid). Oocytes fertilized with MICA-treated (MT) [and thus PDHc/DLD-inhibited] spermatozoa showed defective fertilization where 2nd polar body release and pronuclei formation were not observed. Defective fertilization was attributable to capacitation failure owing to high lactate and low intracellular pH and calcium in MT-spermatozoa during capacitation. Moreover, this defect could be overcome by alkalinizing spermatozoa, before fertilization. Increasing intracellular calcium in spermatozoa pre-IVF and in defectively-fertilized oocytes, post-fertilization rescued the arrest seen, suggesting the role of intracellular calcium from either of the gametes in fertilization. Parallel experiments carried out with control spermatozoa capacitated in medium with low extracellular pH or high lactate substantiated the necessity of optimal sperm intracellular lactate levels, intracellular pH and calcium during sperm capacitation, for proper fertilization. Conclusions This study confirms the importance of pyruvate/lactate metabolism in capacitating spermatozoa for successful fertilization, besides revealing for the first time the importance of sperm PDHc/ DLD in fertilization, via the modulation of sperm intracellular lactate, pH and calcium during capacitation. In addition, the observations made in the IVF studies in hamsters suggest that capacitation failures could be a plausible cause of unsuccessful fertilization encountered during human assisted reproductive technologies, like IVF and ICSI. Our studies indicate a role of sperm capacitation in the post-penetration events during fertilization.

Sailasree, Purnima; Singh, Durgesh K.; Kameshwari, Duvurri B.; Shivaji, Sisinthy

2014-01-01

40

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

PubMed Central

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.

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

2013-01-01

41

Mouse Oocyte Toxicity Assay.  

National Technical Information Service (NTIS)

A standard toxicity assay based on the mouse oocyte system is described. Its application to toxicity determinations of complex mixtures as well as of pure compounds is shown. Because its oocytes are especially sensitive, the juvenile mouse is used. To fac...

J. S. Felton R. L. Dobson

1982-01-01

42

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, ...

43

Effects of fetuin on zona pellucida hardening and fertilizability of equine oocytes matured in vitro.  

PubMed

In vitro fertilization (IVF) has had poor success in the horse, a situation related to low rates of sperm penetration through the zona pellucida (ZP). Zona pellucida hardening (ZPH) is seen in mouse and rat oocytes cultured in serum-free medium. The hardened ZP is refractory to sperm penetration. Fetuin, a component of fetal calf serum, inhibits ZPH and allows normal fertilization rates in oocytes cultured in the absence of serum. We evaluated whether fetuin is present in horse serum and follicular fluid (FF) and whether fetuin could inhibit ZPH in equine oocytes matured in vitro, thus increasing sperm penetration during IVF. The presence of fetuin in equine serum and FF was confirmed by immunoblotting. Oocytes submitted to in vitro maturation (IVM) in medium containing fetuin were used for ZPH assay or IVF. Intracytoplasmic sperm injection (ICSI) was carried out as a control procedure. The presence of fetuin during IVM did not affect the rate of maturation to metaphase II. Maturation of oocytes in the presence of fetuin reduced ZPH in a dose-dependent manner. After both IVF and ICSI, there was no significant difference in oocyte fertilization between fetuin-treated and untreated oocytes. The fertilization rate was significantly higher after ICSI than after IVF, both in fetuin-treated and in untreated oocytes. In conclusion, fetuin reduced ZPH in equine oocytes but did not improve sperm penetration during IVF. This implies that, in the horse, "spontaneous" ZPH is unlikely to be the major factor responsible for inhibiting sperm penetration in vitro. PMID:10411537

Dell'Aquila, M E; De Felici, M; Massari, S; Maritato, F; Minoia, P

1999-08-01

44

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

PubMed

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

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

2014-01-01

45

Human oocyte cryopreservation: new perspectives regarding oocyte survival  

Microsoft Academic Search

The success of human oocyte cryopreservation depends on morphological and biophysical factors that could influence oocyte survival after thawing. Various attempts to cryopreserve human oocytes have been performed with contrasting results. Therefore the effect of some factors, such as the presence or absence of the cumulus oophorus, the sucrose concentration in the freezing solution and the exposure time to cryoprotectants,

R. Fabbri; E. Porcu; T. Marsella; G. Rocchetta; S. Venturoli; C. Flamigni

2001-01-01

46

Liquid penetrants  

NASA Technical Reports Server (NTRS)

Liquid-penetrant inspection is discussed for surface defects in solids. The principle advantages are considered to be its simplicity and economy. The techniques and penetrants are described along with the developers. Commercially available equipment is also described.

Pasley, R. L.

1973-01-01

47

Single Oocyte Bisulfite Mutagenesis  

PubMed Central

Epigenetics encompasses all heritable and reversible modifications to chromatin that alter gene accessibility, and thus are the primary mechanisms for regulating gene transcription1. 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 genes2. 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 level3. Development of the bisulfite mutagenesis assay has advanced from those originally reported4-6 towards ones that are more sensitive and reproducible7. One key advancement was embedding smaller amounts of DNA in an agarose bead, thereby protecting DNA from the harsh bisulfite treatment8. This enabled methylation analysis to be performed on pools of oocytes and blastocyst-stage embryos9. The most sophisticated bisulfite mutagenesis protocol to date is for individual blastocyst-stage embryos10. 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 oocytes11, 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 analysis12. 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.

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

2012-01-01

48

Implications of oocyte cryostorage for the practice of oocyte donation.  

PubMed

As the efficiency of oocyte cryopreservation has increased rapidly in recent years, oocytes are currently being stored either in the course of IVF treatments or as a fertility preservation measure. These practices may have an impact on the number of available donor oocytes due to two different dynamics: first, a certain percentage of women for whom oocytes were cryopreserved will eventually not use their oocytes and may decide to donate them to others; secondly, especially in the practice of social freezing, women may opt to donate a portion of the retrieved oocytes in 'freeze-and-share' schemes in order to reduce the costs. In this article, we aim to sketch the ethical implications of such developments in general and the issue of payment to oocyte donors in particular. PMID:22802093

Mertes, Heidi; Pennings, Guido; Dondorp, Wybo; de Wert, Guido

2012-10-01

49

Decreased expression of CD9 in bovine oocytes after cryopreservation and the relationship to fertilization capacity.  

PubMed

This study was conducted to investigate the effect of vitrification of bovine metaphase-II (MII) oocytes on CD9 expression and fertilization capacity. Surviving vitrified/warmed oocytes were used to detect CD9 distribution (fluorescence microscopy), CD9 mRNA (qRT-PCR), and CD9 protein expression (Western blot), and to analyze in vitro fertilization rates (number of sperm bound to or that penetrated the oocytes) after removing the zona pellucida. Fresh oocytes acted as control. The experimental results showed that the vitrification/warming procedures significantly decreased CD9 expression at the mRNA and protein levels, and changed the CD9 distribution pattern in bovine oocytes. After fertilization in vitro, the average number of sperm binding and penetration of vitrified oocytes were significantly lower than those of the non-vitrified oocytes. In conclusion, vitrification of bovine oocytes caused a decrease in CD9 expression at the mRNA and protein levels, and an alteration of CD9 distribution pattern, which may have resulted in lowered fertilization capacity. PMID:23572435

Zhou, Guang-Bin; Zeng, Yan; Meng, Qing-Gang; Liu, Ying; Dai, Yun-Ping; Zhu, Shi-En; Bunch, Thomas D; Hou, Yun-Peng

2013-06-01

50

Nitric oxide delays oocyte aging.  

PubMed

Nitric oxide (NO) is a ubiquitous signaling molecule that plays a crucial role in oocyte maturation and embryo development. However, its role in oocyte aging is unclear. To examine how NO affects oocyte aging, we retrieved young and relatively old mouse oocytes and exposed them to increasing concentrations of NO donor S-nitroso acetyl penicillamine (SNAP). Aging related phenomena of ooplasmic microtubule dynamics (OMD), cortical granule (CG) exocytosis, zona pellucida (ZP) hardening, and spindle/chromatin integrity were studied at each SNAP concentration using fluorescence immunocytochemistry and confocal microscopy and compared with respective unexposed controls. Exposure of both young and old oocytes to NO resulted in a significant diminution in OMD and ZP dissolution time, whereas spontaneous CG loss decreased in old NO exposed oocytes compared to controls (P < 0.001 for all). Furthermore, NO exposure decreased the rate of spindle abnormalities in oocytes compared to unexposed controls. Interestingly, in old oocytes, the positive influence of NO was attenuated beyond 0.23 microM/min and disappeared at 0.46 microM/min NO. Overall, a significant dose-response relationship was noted between NO exposure and markers of aging with between 50 and 100 microM SNAP (0.11-0.23 microM/min NO, P < 0.0001). Collectively, our results demonstrate for the first time that exposure to NO delays oocyte aging and improves the integrity of the microtubular spindle apparatus in young and old oocytes. PMID:16114873

Goud, Anuradha P; Goud, Pravin T; Diamond, Michael P; Abu-Soud, Husam M

2005-08-30

51

Oocyte freezing: here to stay?  

PubMed

Oocyte freezing is an established technology but, in contrast to embryo freezing, it has very limited application in clinical IVF programmes. Is there a chance that oocyte freezing will become an integrated routine in assisted reproductive technology? The delicate cytological architecture of the oocyte with a cold-sensitive spindle and a hardening zona have made the frozen oocyte 'unwanted' in assisted reproductive technology. Nevertheless, empirical improvements in freezing protocols and the use of ICSI for fertilization have led to an increasing number of live births. This mitigates against a simple ban on oocyte freezing. While efficiency of oocyte freezing can certainly be further improved by basic research, it is clear that there are humanitarian reasons for considering oocyte freezing as a future fully utilized assisted reproductive technology. The storage of the female genome as a particulate entity can provide an alternative in case of moral, ethical, legal or religious concerns about embryo freezing. Oocyte freezing can also offer hope for oocyte donation and preservation of fertility for women facing ovarian loss. The message is one of cautious optimism when looking for a place for oocyte freezing in routine assisted reproductive technology. PMID:14640378

Van der Elst, Josiane

2003-01-01

52

Oocyte freezing: timely reproductive insurance?  

PubMed

Cryopreservation of unfertilised oocytes for later use in initiating pregnancy is now a viable technology, with acceptable pregnancy rates (over 20% per thaw cycle). Oocyte cryopreservation used as a form of insurance against "social" (age-related) infertility can improve the lifetime chance of pregnancy in women who defer pregnancy into their late 30s or early 40s. We report two pregnancies using oocytes that were frozen for social rather than medical reasons, as part of a larger series of nine pregnancies using cryopreserved oocytes. Use of oocytes harvested and frozen from women aged under 35 years may more than double the chance of pregnancy for a 41-year-old woman. The disadvantages of oocyte freezing for social infertility reasons include cost, the usual risks associated with in-vitro fertilisation, and the lack of a guarantee of eventual pregnancy. PMID:19296788

Molloy, David; Hall, Barbara A; Ilbery, Mariannne; Irving, Jacqui; Harrison, Keith L

2009-03-01

53

Oocyte cryopreservation in oncological patients.  

PubMed

The use of chemotherapy and radiotherapy in oncological patients may reduce their reproductive potential. Sperm cryopreservation has been already used in men affected by neoplastic disease. Oocyte cryopreservation might be an important solution for these patients at risk of losing ovarian function. A program of oocyte cryopreservation for oncological patients is also present in our center. From June 1996 to January 2000, 18 patients awaiting chemotherapy and radiotherapy for neoplastic disease were included in our oocyte cryopreservation program. Our experience documents that oocyte storage may be a concrete and pragmatic alternative for oncological patients. The duration of oocyte storage does not seem to interfere with oocyte survival as pregnancies occurred even after several years of gamete cryopreservation in liquid nitrogen. PMID:15041124

Porcu, Eleonora; Fabbri, Raffaella; Damiano, Giuseppe; Fratto, Rosita; Giunchi, Susanna; Venturoli, Stefano

2004-04-01

54

Penetration equations  

SciTech Connect

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.

Young, C.W. [Applied Research Associates, Inc., Albuquerque, NM (United States)

1997-10-01

55

Immunity to Toxoplasmosis in Hamsters.  

National Technical Information Service (NTIS)

Protective immunity to Toxoplasma was studied in hamsters. Immunity developed in 2 to 3 weeks after vaccinations were performed. Vaccination with live RH, T-45, and ts-4 strains afforded the best protection against challenge exposure with the most pathoge...

M. R. Elwell J. K. Frenkel

1984-01-01

56

The transcriptome of human oocytes  

Microsoft Academic Search

The identification of genes and deduced pathways from the mature human oocyte can help us better understand oogenesis, folliculogenesis, fertilization, and embryonic development. Human metaphase II oocytes were used within minutes after removal from the ovary, and its transcriptome was compared with a reference sample consisting of a mixture of total RNA from 10 different normal human tissues not including

Arif Murat Kocabas; Javier Crosby; Hasan H. Otu; Zeki Beyhan; Handan Can; Wai-Leong Tam; Guilherme J. M. Rosa; Robert G. Halgren; Bing Lim; Emilio Fernandez; Jose Bernardo Cibelli

2006-01-01

57

The transcriptome of human oocytes  

PubMed Central

The identification of genes and deduced pathways from the mature human oocyte can help us better understand oogenesis, folliculogenesis, fertilization, and embryonic development. Human metaphase II oocytes were used within minutes after removal from the ovary, and its transcriptome was compared with a reference sample consisting of a mixture of total RNA from 10 different normal human tissues not including the ovary. RNA amplification was performed by using a unique protocol. Affymetrix Human Genome U133 Plus 2.0 GeneChip arrays were used for hybridizations. Compared with reference samples, there were 5,331 transcripts significantly up-regulated and 7,074 transcripts significantly down-regulated in the oocyte. Of the oocyte up-regulated probe sets, 1,430 have unknown function. A core group of 66 transcripts was identified by intersecting significantly up-regulated genes of the human oocyte with those from the mouse oocyte and from human and mouse embryonic stem cells. GeneChip array results were validated using RT-PCR in a selected set of oocyte-specific genes. Within the up-regulated probe sets, the top overrepresented categories were related to RNA and protein metabolism, followed by DNA metabolism and chromatin modification. This report provides a comprehensive expression baseline of genes expressed in in vivo matured human oocytes. Further understanding of the biological role of these genes may expand our knowledge on meiotic cell cycle, fertilization, chromatin remodeling, lineage commitment, pluripotency, tissue regeneration, and morphogenesis.

Kocabas, Arif Murat; Crosby, Javier; Ross, Pablo J.; Otu, Hasan H.; Beyhan, Zeki; Can, Handan; Tam, Wai-Leong; Rosa, Guilherme J. M.; Halgren, Robert G.; Lim, Bing; Fernandez, Emilio; Cibelli, Jose Bernardo

2006-01-01

58

Chromosome transfer in mature oocytes  

PubMed Central

In this article, we describe detailed protocols for the isolation and transfer of spindlechromosomal complexes between mature, metaphase II-arrested oocytes. In brief, the spindlechromosomal 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 karyoplastcytoplast 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.

Tachibana, Masahito; Sparman, Michelle; Mitalipov, Shoukhrat

2011-01-01

59

Comparison of embryonic developmental competence of mouse oocytes grown with and without serum.  

PubMed

The first objective of this study was to determine whether oocyte growth in serum-free medium affects the solubility of the zona pellucida to alpha-chymotrypsin digestion, which is an index of zona pellucida "hardening" and reflects the potential penetrability of the zona pellucida by sperm. Oocyte-granulosa cell complexes were isolated from the preantral follicles of 12-day-old mice and cultured for 10 days in medium containing 5% fetal bovine serum (FBS) or in serum-free medium. The zonae pellucidae of oocytes grown in serum-free medium were four times as hard as freshly isolated germinal vesicle (GV)-stage oocytes grown in vivo or oocytes grown in vitro in FBS-containing medium. The hardening of the zonae pellucidae of oocytes grown in serum-free medium was prevented by addition of fetuin. The second objective was to compare the competence to undergo embryogenesis of oocytes that grew in serum-free vs. FBS-containing medium. Approximately 70% of the oocytes underwent maturation regardless of whether the medium was serum-free or contained FBS. Of the mature ova grown in medium containing FBS, 53% cleaved to the two-cell stage after insemination compared with only 6% of the ova grown in serum-free medium. Addition of fetuin to the serum-free medium used for oocyte growth increased the frequency of cleavage to the two-cell stage. Of the embryos derived from oocytes that grew in FBS-containing medium, 70% completed the two-cell stage to blastocyst transition.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1381197

Eppig, J J; Wigglesowrth, K; O'Brien, M J

1992-05-01

60

Shared oocyte donation: societys benefits  

Microsoft Academic Search

Objective: To assess the efficacy of oocyte donation when a cohort of oocytes is shared between two phenotypically matched recipients.Design: A retrospective analysis of a program using shared anonymous oocyte donation.Setting: Academic infertility center.Patient(s): Recipient women with partial or complete ovarian failure; oocyte donors who have been properly screened.Intervention(s): Each oocyte donor was phenotypically matched with two potential recipients. The

Maureen Moomjy; Robin Mangieri; Fernando Beltramone; Ina Cholst; Lucinda Veeck; Zev Rosenwaks

2000-01-01

61

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

PubMed

IVF in horses is rarely successful. One reason for this could be the failure of sperm to fully capacitate or exhibit hyperactive motility. We hypothesized that the zona pellucida (ZP) of equine oocytes prevents fertilization invitro, and bypassing the ZP would increase fertilization rates. Limited availability of equine oocytes for research has necessitated the use of heterologous oocyte binding assays using bovine oocytes. We sought to validate an assay using bovine oocytes and equine sperm and then to demonstrate that bypassing the ZP using perivitelline sperm injections (PVIs) with equine sperm capacitated with dilauroyl phosphatidylcholine would result in higher fertilization rates than standard IVF in bovine and equine oocytes. In experiment 1, bovine oocytes were used for (1) IVF with bovine sperm, (2) IVF with equine sperm, and (3) intracytoplasmic sperm injections (ICSIs) withequine sperm. Presumptive zygotes were either stained with 4',6-diamidino-2-phenylindole from 18 to 26hours at 2-hour intervals or evaluated for cleavage at 56hours after addition of sperm. Equine sperm fertilized bovine oocytes; however, pronuclei formation was delayed compared with bovine sperm after IVF. The delayed pronuclear formation was not seen after ICSI. In experiment 2, bovine oocytes were assigned to the following five groups: (1) cumulus oocyte complexes (COCs) coincubated with bovine sperm; (2) COC exposed to sucrose then coincubated with bovine sperm; (3) COC coincubated with equine sperm; (4) COC exposed to sucrose, and coincubated with equine sperm; and (5) oocytes exposed to sucrose, and 10 to 15 equine sperm injected into the perivitelline (PV) space. Equine sperm tended (P=0.08) to fertilize more bovine oocytes when injected into the PV space than after IVF. In experiment 3, oocytes were assigned to the following four groups: (1) IVF, equine, and bovine COC coincubated with equine sperm; (2) PVI of equine and bovine oocytes; (3) PVI with equine oocytes pretreated with sucrose; and (4) ICSI of equine oocytes. Oocytes were examined at 24 hours for cleavage. No equine oocytes cleaved after IVF or PVI. However, ICSI conducted with equine sperm treated with dilauroyl phosphatidylcholine resulted in 85% of the oocytes cleaving. Sperm injected into the PV space of equine oocytes did not appear to enter the ooplasm. This study validated the use of bovine oocytes for equine sperm studies and indicates that failure of equine IVF is more than an inability of equine sperm to penetrate the ZP. PMID:24815920

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

2014-07-15

62

Chromosome transfer in mature oocytes  

Microsoft Academic Search

In this article, we describe detailed protocols for the isolation and transfer of spindlechromosomal complexes between mature, metaphase II-arrested oocytes. In brief, the spindlechromosomal 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 karyoplastcytoplast membrane fusion. Newly reconstructed oocytes consist of

Masahito Tachibana; Michelle Sparman; Shoukhrat Mitalipov

2010-01-01

63

Nanoliter droplet vitrification for oocyte cryopreservation  

PubMed Central

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.

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

64

Pregnancy resulting from cattle oocytes matured and fertilized in vitro.  

PubMed

Follicular oocytes (n = 81) collected from cattle at a local slaughterhouse were matured and fertilized in vitro. Of 27 ova 19 (70%) were penetrated by spermatozoa and 40/54 (74%) inseminated ova transferred surgically to the oviducts of a synchronized heifer were recovered by non-surgical flushing of the uterine horns 6 days later. Of the 40 ova 15 (38%) were at the morula, early blastocyst or diminutive morula stages. Culture in vitro sustained further development of all embryos and 9 were expanding or expanded blastocysts. One pregnancy resulted from non-surgical transfer of 2 blastocysts. The results demonstrate that immature oocytes from cattle can be matured and fertilized in vitro, subsequently develop to the blastocyst stage, and develop into a normal pregnancy after non-surgical transfer. PMID:3430467

Xu, K P; Greve, T; Callesen, H; Hyttel, P

1987-11-01

65

An overview of oocyte cryopreservation.  

PubMed

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

Stachecki, James J; Cohen, Jacques

2004-08-01

66

Ultrastructure of in vitro Matured Human Oocytes  

PubMed Central

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

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

2013-01-01

67

Follicle cell regulation of mammalian oocyte growth.  

PubMed

To investigate mechanisms of follicle cell control on mammalian oocyte growth, preantral mouse oocytes free from surrounding follicle cells were individually cocultured with monolayers of different somatic cells competent to form gap junctions, and the rate of in vitro oocyte growth was directly correlated with the level of metabolic coupling on the same cells. The results indicate that 1) at a similar extent of metabolic coupling, mouse oocytes grew on follicle cells but not on 3T3 and Sertoli cell monolayers, and 2) the growth rate of oocytes cultured on follicle cells was dependent on the extent of metabolic coupling. It was concluded that gap-junction-mediated nutrition of ovarian mouse oocytes exerted by somatic cells is necessary but not sufficient to maintain oocyte growth. A specific regulatory role of follicle cells on mammalian oocyte growth is proposed. PMID:3612052

Buccione, R; Cecconi, S; Tatone, C; Mangia, F; Colonna, R

1987-06-01

68

Technical approaches to correction of oocyte aneuploidy  

Microsoft Academic Search

BACKGROUND: This study describes the technical approaches used in treatment of age-related oocyte aneuploidy, the efficiency of each step of nuclear transplantation into mouse and human oocytes, and the ability of germinal vesicle (GV) transplantation to restore artificially induced ooplasmic damage. Finally, it examines the possibility of constructing viable female gametes by transferring diploid somatic cell nuclei into enucleated oocytes.

Gianpiero D. Palermo; Takumi Takeuchi; Zev Rosenwaks

2002-01-01

69

Artificial expression of aquaporin-3 improves the survival of mouse oocytes after cryopreservation.  

PubMed

Successful cryopreservation of mammalian cells requires rapid transport of water and cryoprotective solutes across the plasma membrane. Aquaporin-3 is known as a water/solute channel that can transport water and neutral solutes such as glycerol. In this study we examined whether artificial expression of aquaporin-3 in mouse oocytes can improve water and glycerol permeability and oocyte survival after cryopreservation. Immature mouse oocytes were injected with aquaporin-3 cRNA and were cultured for 12 h. Then the hydraulic conductivity (L(P)) and glycerol permeability (P(GLY)) of matured oocytes were determined from the relative volume changes in 10% glycerol in PB1 medium at 25 degrees C. Mean +/- SD values of L(P) and P(GLY) of cRNA-injected oocytes (3.09 +/- 1.22 micro m min(-1) atm(-1) and 3.69 +/- 1.47 x 10(-3) cm/min, respectively; numbers of oocytes = 25) were significantly higher than those of noninjected oocytes (0.83 +/- 0.02 micro m min(-1) atm(-1) and 0.07 +/- 0.02 x 10(-3) cm/min, respectively; n = 13) and water-injected oocytes (0.87 +/- 0.10 micro m min(-1) atm(-1) and 0.08 +/- 0.02 x 10(-3) cm/min, respectively; n = 20). After cryopreservation in a glycerol-based solution, 74% of cRNA-injected oocytes (n = 27) survived as assessed by their morphological appearance, whereas none of the water-injected oocytes survived (n = 10). When cRNA-injected oocytes that survived cryopreservation were inseminated in vitro, the penetration rate was 40% (n = 48) and the cleavage rate was 31% (n = 70), showing that oocytes retain their ability to be fertilized. This is the first report to show that artificial expression of a water/solute channel in a cell improves its survival after cryopreservation. This approach may enable cryopreservation of cells that have been difficult to cryopreserve. PMID:12493699

Edashige, Keisuke; Yamaji, Yohei; Kleinhans, F W; Kasai, Magosaburo

2003-01-01

70

Comparison and Avoidance of Toxicity of Penetrating Cryoprotectants  

PubMed Central

The objective of this study was to elucidate the toxicity of widely used penetrating cryoprotective agents (CPAs) to mammalian oocytes. To this end, mouse metaphase II (M II) oocytes were exposed to 1.5 M solutions of dimethylsulfoxide (DMSO), ethylene glycol (EG), or propanediol (PROH) prepared in phosphate buffered saline (PBS) containing 10% fetal bovine serum. To address the time- and temperature-dependence of the CPA toxicity, M II oocytes were exposed to the aforementioned CPAs at room temperature (RT, ?23C) and 37C for 15 or 30 minutes. Subsequently, the toxicity of each CPA was evaluated by examining post-exposure survival, fertilization, embryonic development, chromosomal abnormalities, and parthenogenetic activation of treated oocytes. Untreated oocytes served as controls. Exposure of MII oocytes to 1.5 M DMSO or 1.5 M EG at RT for 15 min did not adversely affect any of the evaluated criteria. In contrast, 1.5 M PROH induced a significant increase in oocyte degeneration (54.2%) and parthenogenetic activation (16%) under same conditions. When the CPA exposure was performed at 37C, the toxic effect of PROH further increased, resulting in lower survival (15%) and no fertilization while the toxicity of DMSO and EG was still insignificant. Nevertheless, it was possible to completely avoid the toxicity of PROH by decreasing its concentration to 0.75 M and combining it with 0.75 M DMSO to bring the total CPA concentration to a cryoprotective level. Moreover, combining lower concentrations (i.e., 0.75 M) of PROH and DMSO significantly improved the cryosurvival of MII oocytes compared to the equivalent concentration of DMSO alone. Taken together, our results suggest that from the perspective of CPA toxicity, DMSO and EG are safer to use in slow cooling protocols while a lower concentration of PROH can be combined with another CPA to avoid its toxicity and to improve the cryosurvival as well.

Szurek, Edyta A.; Eroglu, Ali

2011-01-01

71

Comparative carcinogenesis by nitrosomethylalkylamines in Syrian hamsters.  

PubMed

Six homologous nitrosomethyl-n-alkylamines, from n-propyl (C-3) to n-octyl (C-8), were administered by gavage to groups of 12 male and 12 female Syrian golden hamsters as solutions in corn oil:ethyl acetate (2:1). The solutions of C-8 to C-4 were equimolar; nitrosomethyl-n-butylamine (C-4) and nitrosomethyl-n-propylamine (C-3) were given at a lower concentration. Treatment with 0.2 ml of solution lasted 23 to 50 wk, being stopped when several hamsters had died. Additional groups of hamsters were treated similarly with nitrosomethylaniline and nitrosomethylcyclohexylamine. Excepting hamsters given the latter, treated animals had reduced survival compared with controls. The incidence of tumors in hamsters given nitrosomethylaniline and nitrosomethylcyclohexylamine was low and occurred in the liver, lungs, and spleen. Hamsters treated with nitrosomethyl-n-propylamine and nitrosomethyl-n-butylamine suffered the greatest decrease in survival. Potency judged by this criterion decreased as the size of the molecule increased in the homologous series. Virtually all of these treated hamsters died with tumors not seen in controls. These tumors, which were common in hamsters given all of the nitrosomethyl-n-alkylamines, were in the liver, lung, forestomach, and nasal mucosa, but the incidences varied somewhat between the compounds and between sexes. Bladder tumors were seen only in hamsters given nitrosamines containing even numbers of carbon atoms in the chain, namely, nitrosomethyl-n-hexylamine and nitrosomethyl-n-octylamine. PMID:3180074

Lijinsky, W; Kovatch, R M

1988-12-01

72

Fate of fertilized human oocytes  

Microsoft Academic Search

Establishing the proportion of fertilized oocytes and early human embryos that proceed to term may help policy makers in their evaluation of when the life of a new human individual begins and in determining the nature of protection to be accorded to it. The rate of spontaneous abortions, although increasing with age, overall does not exceed 15%. However, abortion rates

Giuseppe Benagiano; Manuela Farris; Gedis Grudzinskas

2010-01-01

73

Acetylcholine receptors in human oocytes.  

PubMed

Neurotransmitter receptors have been studied by conventional electrophysiological techniques in the membrane of human ovarian oocytes isolated from ovarian fragments obtained from pre-menopausal women undergoing abdominal surgery for gynaecological conditions. Ovarian oocytes respond to acetylcholine (ACh) concentrations as low as 10(-10) M by hyperpolarizing the membrane and by concomitantly increasing input resistance, in a dose-dependent manner. The response lasts as long as the transmitter is present in the extracellular fluid. No response is elicited by ionophoretically applied ACh. The ACh response has an apparent latency of less than 1 s and a reversal potential of about -12 mV. The response to ACh (10(-8) - 10(-3) M) is unaffected by curare (10(-5) - 10(-4) g/ml) and is blocked by atropine (10(-6) - 10(-4) g/ml). This indicates that ACh receptors in the human oocyte membrane are probably muscarinic in nature. No response is elicited by the amino acids glutamate, aspartate and glycine (up to 10(-3) M), or by noradrenaline, adrenaline and 5-hydroxytryptamine (up to 10(-3) M). On the basis of analogies to the response elicited by agents which activate parthenogenetic development in the oocytes of other mammals, it is suggested that the sperm-carried ACh might be involved in activation processes triggered by sperm-egg interaction. PMID:6699777

Eusebi, F; Pasetto, N; Siracusa, G

1984-01-01

74

Recent Progress in Cryopreservation of Bovine Oocytes  

PubMed Central

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 ?-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.

Hochi, Shinichi

2014-01-01

75

Recent progress in cryopreservation of bovine oocytes.  

PubMed

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 ?-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

Hwang, In-Sul; Hochi, Shinichi

2014-01-01

76

[Study and applications of human oocyte cryopreservation].  

PubMed

The oocyte cryopreservation is more promising than embryo freezing in clinical applications, preservation of female fertility,legal and ethical aspects in this decade. The redundant oocytes retrieved from IVF cycles can potentially donate oocytes, which have unique advantages in economy and feasibility. We have achieved success in embryo freezing, but just obtained poorer results in oocyte cryopreservation which was mainly because of the low rates of survival, fertilization, and cleavage. The character of the plasma membrane and the time of cortical granules present at the metaphase of meiosis II with the spindle system consist of the major difference between oocytes and embryos. Moreover, the oocytes should be fertilized by sperm at the appropriate time. We used a refined slow freezing method to improve the survival rate and increased sucrose concentration to dehydrate oocytes. Vitrification was another approach to prevent harms. Besides, intracytoplasmic sperm injection was used to overcome possible zona hardening after the release of cortical granules. Oocytes after cryopreservation showed serious disturbances of the microtubules immediately after thawing as they were vulnerable to the thermal changes and easy to depolymerize. Fertilization of these oocytes with disorganized spindles led to chromosomal aneuploidy, digyny, and degradation of the cleavage rate. We can improve cryopreserved oocytes to normal fertilization and development by appropriate incubation and timing of insemination, as the microtubules repolymerize in a time dependent way after incubation which is compatible with recovery of the spindles. With the improvement of survival, fertilization, and development, oocyte cryopreservation will play an imperative role. PMID:15605106

Li, Xiao-hong

2004-12-01

77

Cryopreservation of immature and mature human oocytes.  

PubMed

Cryopreservation of oocytes facilitates the long-term storage of oocytes for patients in danger of losing ovarian function. It also alleviates many of the ethical concerns associated with embryo cryopreservation. Problems associated with metaphase II oocyte cryopreservation include zona pellucida hardening and spindle damage. The cryopreservation of germinal vesicle-stage oocytes has been undertaken as a means of circumventing the problem of spindle damage in mature oocytes. One of the main disadvantages of immature oocyte cryopreservation is the fact that in vitro maturation is required post-thaw. The majority of live births from oocyte cryopreservation have involved the use of 1,2-propanediol and slow freezing protocols. Various methods have been used in an attempt to improve survival rates. These include vitrification and use of novel cryopreservatives. Future areas of concentration should include in vitro maturation, vitrification, and alternate cryopreservatives. PMID:11941534

Wininger, J D; Kort, H I

2002-02-01

78

Use of frozen-thawed oocytes for efficient production of normal offspring from cryopreserved mouse spermatozoa showing low fertility.  

PubMed

Freezing of spermatozoa and unfertilized oocytes is a useful tool for the conservation of mouse genetic resources. However, the proportion of frozen-thawed oocytes fertilized with spermatozoa in vitro is low because spermatozoa, especially those frozen-thawed, can not penetrate into oocytes because of hardening of the zona pellucida following premature release of cortical granules. To produce offspring efficiently from cryopreserved transgenic mouse gametes, we fertilized frozen-thawed gametes by using intracytoplasmic sperm injection (ICSI) and assessed pre- and postimplantation development of embryos. Compared with fresh unfertilized oocytes, frozen-thawed unfertilized oocytes were highly tolerant to damage by injection, as the survival rates after injection of frozen spermatozoa were 51 and 78%, respectively. Frozen-thawed oocytes that survived after sperm injection developed normally to the blastocyst stage and gave rise to offspring. Moreover, offspring with transgenes also were obtained from frozen gametes fertilized by ICSI. These results demonstrate that ICSI is an efficient technique for producing offspring from transgenic spermatozoa showing low fertility and that use of frozen-thawed oocytes leads to conservation of genetic resources because suboptimally preserved gametes are not wasted. PMID:15884774

Sakamoto, Wataru; Kaneko, Takehito; Nakagata, Naomi

2005-04-01

79

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

PubMed

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

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

2013-09-01

80

Surgical Oocyte Retrieval (SOR): a Method for Collecting Mature Mouse Oocytes Without Euthanasia  

PubMed Central

A novel surgical method for collecting oocytes from unique and irreplaceable mice is described. This method, surgical oocyte retrieval (SOR), facilitates the collection of ovulated oocytes, does not require euthanasia, and preserves reproductive potential. The surgery involves a small incision in the ampulla region of the oviduct, through which the cumulus oocyte mass is removed with a gel-loading pipette. The incision then is closed by using a tissue adhesive, which is required to ensure healing of the incision and containment of any oocytes ovulated after SOR. Two anesthetics, isoflurane and tribromoethanol, were compared for oocyte toxicity during SOR. More dead oocytes were recovered when tribromoethanol was used than when isoflurane was used. Combining SOR and traditional oocyte collection methods yielded more oocytes per BALB/cByJ than did traditional methods alone (41 versus 28 oocytes, respectively). Oocytes collected by using SOR were fertilized and subsequent embryos developed to term comparable to controls. This technique provides an alternative method for oocyte collection and will be valuable for maximizing the number of oocytes from irreplaceable mice.

Byers, Shannon L; Wiles, Michael V; Taft, Robert A

2009-01-01

81

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...

82

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

PubMed

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

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

2014-01-01

83

In vitro fertilization and polyspermy in the pig: Factors affecting fertilization rates and cytoskeletal reorganization of the oocyte  

Microsoft Academic Search

Polyspermy is a common phenomenon in the pig. Extensive information has become available from in vitro studies on not only the quality of oocytes but also the quality of spermatozoa. However, little information is available on the relative penetration rates of fresh and frozen spermatozoa from the same ejaculate from boars of different breeds. The present results, based on a

Hiroyuki Suzuki; Yosuke Saito; Noriko Kagawa; Xiangzhong Yang

2003-01-01

84

Effects of caffeine and casein phosphopeptides on fertilization in vitro of pig oocytes matured in culture.  

PubMed

Two experiments were conducted to assess the effects of caffeine and casein phosphopeptides (CPPs). One experiment tested the ability of frozen-thawed epididymal spermatozoa from boar (A, B, C), of proven low in vitro fertilization rates, to penetrate pig follicular oocytes. The other experiment tested the ability of ejaculated spermatozoa to uptake Ca2+. In Experiment 1, oocytes matured in vitro were inseminated with spermatozoa (Boar A) in medium that contained 0, 2, 5, 10, 15, and 20 mM caffeine and CPPs (1 mg/ml), or in medium that contained the same caffeine concentrations without CPPs. When CPPs were added to the caffeine-containing medium, significantly higher penetration rates were obtained than when the oocytes were inseminated in the CPPs-free medium. When the oocytes were inseminated with the spermatozoa (Boar A, B, C) in medium that contained 5 mM caffeine and dephosphorylated CPPs (dCPP:1 mg/ml), the penetration rate was significantly lower than when the oocytes were inseminated with the spermatozoa in medium containing 5 mM caffeine and CPPs (1 mg/ml). In Experiment 2, the concentration of Ca2+ in ejaculated spermatozoa of proven low in vitro fertilization rates during incubation in the fertilization medium was determined with fluorescence, Fura2/AM. When the medium contained CPPs, the intracellular concentration of Ca2+ in spermatozoa increased with a peak of 113 nM after 90 min of incubation. The concentration of Ca2+ was gradually decreased in the medium without CPPs. However, addition of CPPs in the medium had no effect on the motility of spermatozoa in Experiments 1 and 2.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8011329

Nagai, T; Takenaka, A; Mori, T; Hirayama, M

1994-04-01

85

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

PubMed

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

Lunn, Matthew O; Wright, Shirley J

2013-09-15

86

The role of the oocyte in folliculogenesis.  

PubMed

Novel regulatory proteins have been identified within oocytes that are crucially involved in folliculogenesis. One of the most exciting oocyte signaling molecules is a novel member of the transforming growth factor beta (TGF-beta) superfamily, growth differentiation factor 9 (GDF-9). Loss-of-function studies have established that GDF-9 is obligatory for proper folliculogenesis and fertility in female mice. The current challenges are to understand how oocyte morphogens regulate folliculogenesis and how their actions and interactions are integrated into the overall processes of physiology and pathophysiology. Who would have thought that oocyte morphogens would be so crucial for reproduction? PMID:10856922

Erickson, G F; Shimasaki, S

2000-07-01

87

Fourier analysis of mitochondrial distribution in oocytes  

NASA Astrophysics Data System (ADS)

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.

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

2011-02-01

88

Theoretical and experimental basis of oocyte vitrification.  

PubMed

In the last decades significant advances have been made in successful cryopreservation of mammalian oocytes. Human oocyte cryopreservation has practical application in preserving fertility for individuals at risk of compromised egg quality due to cancer treatments or advanced maternal age. While oocyte cryopreservation success has increased over time, there is still room for improvement. Oocytes are susceptible to cryodamage; which collectively entails cellular damage caused by mechanical, chemical or thermal forces during the vitrification and warming process. This review will delineate many of the oocyte intracellular and extracellular structures that are/may be stressed and/or compromised during cryopreservation. This will be followed by a discussion of the theoretical basis of oocyte vitrification and warming, and a non-exhaustive review of current experimental data and clinical expectations of oocyte vitrification will be presented. Finally, a forward-thinking vision of a potential means of modifying and improving vitrification and warming procedures and success will be proposed. This review addresses theoretical and experimental evidence accumulated over the last two decades supporting the application of vitrification and warming to oocyte cryopreservation. Issues ranging from clinical needs for oocyte cryopreservation, cryopreservation-induced stresses and normal oocyte function, practical application of vitrification-warming of oocytes, and potential future directions will be discussed. In addition, we debate commonly discussed technical methods of oocyte vitrification-warming that may not necessarily be grounded in scientific knowledge. Instead these methodologies are many times theoretical, potentially empirical and commonly lack significant testing and scientific rigor. Questions include: (i) what is the best cryoprotectant? (ii) are some cryoprotectants more toxic compared with others? (iii) how should cryosolutions be mixed with cells? (iv) is there a best container for vitrification? (v) is there a threshold cooling-warming rate or is a faster rate always better? and finally (vi) should oocytes be vitrified with or without adjacent cells? With this said, it is recognized that important advancements have been made in the past decade in oocyte cryopreservation, many times through empirical findings. Finally, we propose some new areas of research that may influence future success of oocyte vitrification and warming, fully recognizing that these theories require mechanical and biological experimental testing. PMID:21763203

Smith, Gary D; Motta, Eduardo E; Serafini, Paulo

2011-09-01

89

Oocyte maturation and IVF in cattle  

Microsoft Academic Search

There is still a clear difference between ova obtained from in vivo maturation and oocytes matured in culture. Eggs obtained following the LH surge in vivo show a higher potential for development. These observations indicate that cytoplasmic competence must be different between the in vitro and the in vivo matured oocytes. Besides these functional differences, a few studies have revealed

M. A. Sirard; P. Blondin

1996-01-01

90

Polycystic ovary syndrome and oocyte developmental competence.  

PubMed

Folliculogenesis is a complex process, in which multiple endocrine and intraovarian paracrine interactions create a changing intrafollicular microenvironment for appropriate oocyte development. Within this microenvironment, bidirectional cumulus cell-oocyte signaling governs the gradual acquisition of developmental competence by the oocyte, defined as the ability of the oocyte to complete meiosis and undergo fertilization, embryogenesis, and term development. These regulatory mechanisms of follicle growth, controlled in part by the oocyte itself, are susceptible to derangement in polycystic ovary syndrome (PCOS), a heterogeneous syndrome characterized by ovarian hyperandrogenism, insulin resistance, and paracrine dysregulation of follicle development. Consequently, only a subset of PCOS patients experience reduced pregnancy outcome after ovarian stimulation for in vitro fertilization. Recent data implicate functional associations between endocrine/paracrine abnormalities, metabolic dysfunction, and altered oocyte gene expression with impaired oocyte developmental competence in women with PCOS. Therefore, an understanding of how developmentally relevant endocrine/paracrine factors interact to promote optimal oocyte developmental is crucial to identify those PCOS patients who might benefit from long-term correction of follicle growth to improve fertility, optimize follicular responsiveness to gonadotropin therapy, and enhance pregnancy outcome by in vitro fertilization. PMID:18081939

Dumesic, Daniel A; Padmanabhan, Vasantha; Abbott, David H

2008-01-01

91

Spindle Dynamics during Meiosis in Drosophila Oocytes  

Microsoft Academic Search

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 previ- ously. We report here the use of the Nonclaret disjunc- tional (Ncd) microtubule motor protein fused to the green fluorescent protein (GFP) to monitor changes in the meiotic spindle

Sharyn A. Endow; Donald J. Komma

1997-01-01

92

Cytoplasmic transfer in oocytes: biochemical aspects  

Microsoft Academic Search

Cytoplasmic control of preimplantation development is not a 'new' concept; the first cytoplasmic transfer experiment was performed in the mouse during the early 1980s, as a means of overcoming cleavage arrest at the 2-cell stage, the '2-cell block'. Since the first human pregnancy following the transfer of cytoplasm from donor oocytes into the oocytes of a patient with a history

Rachel Levy; Kay Elder; Yves Menezo

2004-01-01

93

In vitro Culture of Postimplantation Hamster Embryos.  

National Technical Information Service (NTIS)

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 embryot...

M. T. Ebron-McCoy P. E. Beyer L. A. Oglesby R. J. Kavlock

1988-01-01

94

Directed Student Inquiry: Modeling in Roborovsky Hamsters  

NSDL National Science Digital Library

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 data collection. Based on the premise that this is a directed rather than dictated student inquiry, the activity will vary based on discussions and recommendations suggested by each class.

Bouchard, Adam; Elwess, Nancy L.

2007-04-01

95

Cryopreservation of human oocytes and ovarian tissue.  

PubMed

Oocyte cryopreservation has the potential to be an important adjunct to assisted reproductive technologies and bypasses some ethical, moral, and religious dilemmas posed by human embryo cryopreservation. The success of human oocyte cryopreservation depends on morphological and biophysical factors that could influence oocyte survival after thawing. Among the morphological factors, the maturity, quality, size of the oocyte, the presence or the absence of the cumulus oophorus seems to play an important role in oocyte survival after thawing. The main biophysical factor of cellular disruption during cryopreservation process in the intracellular ice formation that can be avoided by an adequate cell dehydration; thus reducing the intracellular water by increasing the dehydration process we can limit the damages of the cryopreservation procedure. The dehydration process can be affected by the presence and concentration of the cryoprotectants in the freezing solutions (equilibration and loading solutions), and by the freezing and thawing rate. Two additional properties of cryoprotectants help to protect cells during slow cooling, when the cells are very dehydrated and are surrounded by concentrated salts. The cryoprotectants appear to reduce damage caused by high levels of salt, a property known as salt buffering. Some events occurring to the oocyte during cryopreservation procedure has been found to be a premature exocitosis of cortical granules, leading to an intempestive zona hardening and consequently to a reduction of fertilization rate, and the cryoinjury to the zona pellucida leading to a polispermic fertilization. ICSI is an efficient method to by pass these two events and to achieve a satisfactory outcome in terms of normal fertilization of cryopreserved oocytes. The application of the ICSI to cryopreserved oocytes did not seem to increase the degeneration rate after insemination with respect to fresh oocytes. The increased oocyte survival rate and the use of ICSI have facilitated the recent increase in the number of pregnancies and live birth. PMID:16732414

Fabbri, Raffaella

2006-01-01

96

Fluorescent Penetrant Inspection.  

National Technical Information Service (NTIS)

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 su...

S. Sastri

1990-01-01

97

Penetration of concrete targets  

SciTech Connect

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.

Forrestal, M.J. [Sandia National Labs., Albuquerque, NM (United States); Cargile, J.D. [Corps of Engineers, Vicksburg, MS (United States). Waterways Experiment Station; Tzou, R.D.Y. [New Mexico Univ., Albuquerque, NM (United States). Dept. of Mechanical Engineering

1993-08-01

98

Hard Rock Penetration Technology.  

National Technical Information Service (NTIS)

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 Overvi...

2005-01-01

99

Current trends and progress in clinical applications of oocyte cryopreservation  

PubMed Central

Purpose of review To delineate the current trends in the clinical application of oocyte cryopreservation. Recent findings Although the first live birth from oocyte cryopreservation was reported approximately three decades ago, significant improvement in the clinical application of oocyte cryopreservation took place only over the past decade. On the basis of the available evidence suggesting that success rates with donor oocyte vitrification are similar to that of IVF with fresh donor oocytes, the American Society of Reproductive Medicine has recently stated that oocyte cryopreservation should no longer be considered experimental for medical indications, outlying elective oocyte cryopreservation. Meanwhile, a few surveys on the attitudes toward oocyte cryopreservation revealed that elective use for the postponement of fertility is currently the most common indication for oocyte cryopreservation. Most recently, a randomized controlled trial revealed important evidence on the safety of nondonor oocyte cryopreservation, and confirmed that the clinical success of vitrification is comparable to that of IVF with fresh oocytes. Summary The evidence suggesting similar IVF success rates with both donor and nondonor cryopreserved oocytes compared with fresh oocytes will increase the utilization of elective oocyte cryopreservation. Appropriate counseling of women for oocyte cryopreservation requires the establishment of age-based clinical success rates with cryopreserved oocytes for various indications.

Cil, Aylin P.; Seli, Emre

2013-01-01

100

Oocyte ageing and its cellular basis.  

PubMed

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

Eichenlaub-Ritter, Ursula

2012-01-01

101

Abnormal chromosomal arrangements in human oocytes.  

PubMed

Ninety-one human oocytes, lacking signs of fertilization 50 h after insemination in vitro, were investigated cytogenetically to assess the frequency and type of chromosomal abnormalities. Chromosome spreading permitted adequate karyotyping in 55 oocytes. Non-determined numerical aberrations occurred with the following frequencies: hypohaploidy, 10.9% (6/55), hyperhaploidy, 14.5% (8/55) and hyperdiploidy, 3.6% (2/55). Total aneuploidy occurred with a frequency of 29.1% and was observed in oocytes from 30 patients. No correlation was found between specific chromosomal aberrations and type of infertility, stimulation treatment or gonadotrophin levels. On the other hand, the frequency of aneuploidy was significantly higher (P less than 0.05) in patients greater than 35 years of age. Two chromosomal complements (3.6%) had structural rearrangements; one oocyte had both structural and numerical chromosomal abnormalities and the other had differently condensed regions on the long arms of three chromosomes from group C. The overall frequency of chromosomal aberrations was 32.7%. Only two samples contained an additional set of polar body chromosomes. Thirteen oocytes presented sperm chromosomes in an arrested stage of premature chromosome condensation of the G1 phase and four oocytes showed asynchronous condensation of pronuclear chromosomes. Finally, it was concluded that the high proportion of chromosomal aberrations observed in human oocytes may contribute significantly to abnormal embryonic development in vitro. PMID:2254403

Macas, E; Floersheim, Y; Hotz, E; Imthurn, B; Keller, P J; Walt, H

1990-08-01

102

ACOG: Committee Opinion No. 584: oocyte cryopreservation.  

PubMed

: In 2013, the American Society for Reproductive Medicine and the Society for Assisted Reproductive Technology published a joint document, Mature Oocyte Cryopreservation: A Guideline, which addresses advances in techniques to freeze human eggs that have resulted in significant recent improvements in pregnancy success. Based on the current state of evidence, modern procedures to cryopreserve oocytes should no longer be considered experimental. The American College of Obstetricians and Gynecologists' Committee on Gynecologic Practice endorses the joint document and encourages its use by Fellows. There are not yet sufficient data to recommend oocyte cryopreservation for the sole purpose of circumventing reproductive aging in healthy women. PMID:24463693

2014-01-01

103

Fluorescent penetrant inspection  

NASA Technical Reports Server (NTRS)

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.

Sastri, Sankar

1990-01-01

104

The Hamster Polyomavirusa Brief Review of Recent Knowledge  

Microsoft Academic Search

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

Siegfried Scherneck; Rainer Ulrich; Jean Feunteun

2001-01-01

105

Assisted oocyte activation following ICSI fertilization failure.  

PubMed

The capacity of intracytoplasmic sperm injection (ICSI) to permit almost any type of spermatozoa to fertilize oocytes has made it the most successful treatment for male factor infertility. Despite its high success rates, fertilization failure following ICSI still occurs in 1-3% of couples. Assisted oocyte activation (AOA) is being increasingly applied in human assisted reproduction to restore fertilization and pregnancy rates in couples with a history of ICSI fertilization failure. However, controversy still exists mainly because the artificial activating agents do not mimic precisely the initial physiological processes of mammalian oocyte activation, which has led to safety concerns. This review addresses the mechanism of human oocyte activation and the relatively rare phenomenon of fertilization failure after ICSI. Next, it describes the current diagnostic approaches and focuses on the application, efficiency and safety of AOA in human assisted reproduction. PMID:24656559

Vanden Meerschaut, Frauke; Nikiforaki, Dimitra; Heindryckx, Bjrn; De Sutter, Petra

2014-05-01

106

Mitochondrial functions on oocytes and preimplantation embryos  

Microsoft Academic Search

Oocyte quality has long been considered as a main limiting factor for in vitro fertilization (IVF). In the past decade, extensive\\u000a observations demonstrated that the mitochondrion plays a vital role in the oocyte cytoplasm, for it can provide adenosine\\u000a triphosphate (ATP) for fertilization and preimplantation embryo development and also act as stores of intracellular calcium\\u000a and proapoptotic factors. During the

Li-ya Wang; Da-hui Wang; Xiang-yang Zou; Chen-ming Xu

2009-01-01

107

Effects of Resveratrol on Vitrified Porcine Oocytes  

PubMed Central

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.

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

2013-01-01

108

Ultrastructure of human mature oocytes after vitrification  

PubMed Central

Since the introduction of human assisted reproduction, oocyte cryopreservation has been regarded as an attractive option to capitalize the reproductive potential of surplus oocytes and preserve female fertility. However, for two decades the endeavor to store oocytes has been limited by the not yet optimized methodologies, with the consequence of poor clinical outcome or of uncertain reproducibility. Vitrification has been developed as the promising technology of cryopreservation even if slow freezing remains a suitable choice. Nevertheless, the insufficiency of clinical and correlated multidisciplinary data is still stirring controversy on the impact of this technique on oocyte integrity. Morphological studies may actually provide a great insight in this debate. Phase contrast microscopy and other light microscopy techniques, including cytochemistry, provided substantial morpho-functional data on cryopreserved oocyte, but are unable to unraveling fine structural changes. The ultrastructural damage is one of the most adverse events associated with cryopreservation, as an effect of cryo-protectant toxicity, ice crystal formation and osmotic stress. Surprisingly, transmission electron microsco py has attracted only limited attention in the field of cryopreservation. In this review, the subcellular structure of human mature oocytes following vitrification is discussed at the light of most relevant ultrastructural studies.

Khalili, M.A.; Maione, M.; Palmerini, M.G.; Bianchi, S.; Macchiarelli, G.; Nottola, S.A.

2012-01-01

109

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

PubMed Central

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.

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

2013-01-01

110

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

PubMed

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 G2-M1 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

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

2013-01-01

111

Revisiting oocyte-somatic cell interactions: in search of novel intrafollicular predictors and regulators of oocyte developmental competence  

PubMed Central

Prediction and improvement of oocyte competence are two critical issues in assisted reproductive technology to improve infertility therapy. The lack of reliable and objective predictors of oocyte developmental competence for oocyte/embryo selection during in vitro fertilization hampers the effectiveness of this technology. Likewise, the low pregnancy rate resulting from in vitro maturation of human oocytes represents a major obstacle for its clinical application. Oocyte competence is progressively acquired during follicular development, and the oocyte plays a dominant role in regulating granulosa cell functions and maintaining the microenvironment appropriate for the development of its competence. Hence, granulosa cell functions are reflective of oocyte competence, and molecular markers of granulosa cells are potentially reliable predictors of oocyte quality. With the advent of the functional genomics era, the transcriptome of granulosa cells has been extensively characterized. Experimental data supporting granulosa cell markers as predictors of oocyte competence are now emerging in both animal models and humans. Future efforts should focus on integrating granulosa cell genetic markers as parameters for oocyte/embryo selection. Moreover, novel in vitro evidence highlights the effectiveness of exogenous oocyte-secreted factors in promoting oocyte developmental competence in animal models. The challenge in evaluating the effect of oocyte-secreted factors on oocyte quality in a clinical setting is to standardize the various preparations of these recombinant proteins and decipher their complex interactions/cooperativity within the germline-somatic cell regulatory loop.

Li, Qinglei; McKenzie, Laurie J.; Matzuk, Martin M.

2008-01-01

112

Caspase 9 is constitutively activated in mouse oocytes and plays a key role in oocyte elimination during meiotic prophase progression  

PubMed Central

In many mammalian species, more than half of the initial oocyte population is eliminated by neonatal life, thus limiting the oocyte reserve for reproduction. The cause or mechanism of this major oocyte loss remains poorly understood. We examined the apoptotic pathway involved in oocyte elimination in wild-type mouse ovaries as well as in Msh5 ?/? ovaries, in which all oocytes were eliminated due to a lack of double strand break repair. Immunoblot and immunofluorescence staining showed that an initiator caspase 9 and an effector caspase 7 were constitutively activated in almost all oocytes in fetal ovaries regardless of their genotypes. In caspase 9 ?/? ovaries, the total number of oocytes remained high while that in wild-type ovaries steadily declined during ovarian development. Therefore, the activation of caspase 9 was required for but did not immediately lead to oocyte demise. We found that XIAP, an endogenous inhibitor of apoptosis, was also abundant in oocytes during meiotic prophase progression. On the other hand, a cleaved form of PARP1, a target of effector caspases, was localized to the nuclei of a limited number of oocytes, and the frequency of cleaved PARP1-positive oocyte nuclei increased significantly higher before all oocytes disappeared in Msh5 ?/? ovaries. We conclude that the mitochondrial apoptotic pathway mediated by caspase 9 is constitutively activated in oocytes and renders the elimination of oocytes with meiotic errors, which can be captured by the cleavage of PARP1.

Ene, Adriana C.; Park, Stephanie; Edelmann, Winfried; Taketo, Teruko

2013-01-01

113

Caspase 9 is constitutively activated in mouse oocytes and plays a key role in oocyte elimination during meiotic prophase progression.  

PubMed

In many mammalian species, more than half of the initial oocyte population is eliminated by neonatal life, thus limiting the oocyte reserve for reproduction. The cause or mechanism of this major oocyte loss remains poorly understood. We examined the apoptotic pathway involved in oocyte elimination in wild-type mouse ovaries as well as in Msh5 -/- ovaries, in which all oocytes were eliminated due to a lack of double strand break repair. Immunoblot and immunofluorescence staining showed that an initiator caspase 9 and an effector caspase 7 were constitutively activated in almost all oocytes in fetal ovaries regardless of their genotypes. In caspase 9 -/- ovaries, the total number of oocytes remained high while that in wild-type ovaries steadily declined during ovarian development. Therefore, the activation of caspase 9 was required for but did not immediately lead to oocyte demise. We found that XIAP, an endogenous inhibitor of apoptosis, was also abundant in oocytes during meiotic prophase progression. On the other hand, a cleaved form of PARP1, a target of effector caspases, was localized to the nuclei of a limited number of oocytes, and the frequency of cleaved PARP1-positive oocyte nuclei increased significantly higher before all oocytes disappeared in Msh5 -/- ovaries. We conclude that the mitochondrial apoptotic pathway mediated by caspase 9 is constitutively activated in oocytes and renders the elimination of oocytes with meiotic errors, which can be captured by the cleavage of PARP1. PMID:23384561

Ene, Adriana C; Park, Stephanie; Edelmann, Winfried; Taketo, Teruko

2013-05-01

114

Congenital Transmission of Experimental Leishmaniasis in a Hamster Model  

PubMed Central

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

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

2012-01-01

115

Congenital transmission of experimental leishmaniasis in a hamster model.  

PubMed

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

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

2012-05-01

116

Mitogenomes of Polar Bodies and Corresponding Oocytes  

PubMed Central

The objective of the present study was to develop an approach that could assess the chromosomal status and the mitochondrial DNA (mtDNA) content of oocytes and their corresponding polar bodies (PBs) with the goal of obtaining a comparative picture of the segregation process both for nuclear and mtDNA. After Whole Genome Amplification (WGA), sequencing of the whole mitochondrial genome was attempted to analyze the segregation of mutant and wild-type mtDNA during human meiosis. Three triads, composed of oocyte and corresponding PBs, were analyzed and their chromosome status was successfully assessed. The complete mitochondrial genome (mitogenome) was almost entirely sequenced in the oocytes (95.99% compared to 98.43% in blood), while the percentage of sequences obtained in the corresponding PB1 and PB2 was lower (69.70% and 69.04% respectively). The comparison with the mtDNA sequence in blood revealed no changes in the D-loop region for any of the cells of each triad. In the coding region of blood mtDNA and oocyte mtDNA sequences showed full correspondence, whereas all PBs had at least one change with respect to the blood-oocyte pairs. In all, 9 changes were found, either in PB1 or PB2: 4 in MT-ND5, 2 in MT-RNR2, and 1 each in MT-ATP8, MT-ND4, MT-CYTB. The full concordance between oocyte and blood in the 3 triads, and the relegation of changes to PBs, revealed the unexpected coexistence of different variants, giving a refined estimation of mitochondrial heteroplasmy. Should these findings be confirmed by additional data, an active mechanism could be postulated in the oocyte to preserve a condition of normality.

Gianoarli, Luca; Luiselli, Donata; Crivello, Anna Maria; Lang, Martin; Ferraretti, Anna Pia; De Fanti, Sara; Magli, M. Cristina; Romeo, Giovanni

2014-01-01

117

Nuclear structures in Tribolium castaneum oocytes.  

PubMed

The first ultrastructural and immunomorphological characteristics of the karyosphere (karyosome) and extrachromosomal nuclear bodies in the red flour beetle, Tribolium castaneum, are presented. The karyosphere forms early in the diplotene stage of meiotic prophase by the gathering of all oocyte chromosomes in a limited nuclear volume. Using the BrUTP assay, T. castaneum oocyte chromosomes united in the karyosphere maintain their transcriptional activity until the end of oocyte growth. Hyperphosphorylated RNA polymerase II and basal transcription factors (TFIID and TFIIH) were detected in the perichromatin region of the karyosphere. The T. castaneum karyosphere has an extrachromosomal capsule that separates chromosomes from the rest of the nucleoplasm. Certain structural proteins (F-actin, lamin B) were found in the capsule. Unexpectedly, the karyosphere capsule in T. castaneum oocytes was found to be enriched in TMG-capped snRNAs, which suggests that the capsule is not only a structural support for the karyosphere, but may be involved in biogenesis of snRNPs. We also identified the counterparts of 'universal' extrachromosomal nuclear domains, Cajal bodies (CBs) and interchromatin granule clusters (IGCs). Nuclear bodies containing IGC marker protein SC35 display some features unusual for typical IGCs. SC35 domains in T. castaneum oocytes are predominantly fibrillar complex bodies that do not contain trimethyl guanosine (TMG)-capped small nuclear (sn) RNAs. Microinjections of 2'-O-methyl (U)22 probes into the oocytes allowed revealing poly(A)+ RNAs in these nuclear domains. Several proteins related to mRNA export (heterogeneous ribonucleoprotein core protein A1, export adapters Y14 and Aly and export receptor NXF1) were also detected there. We believe that unusual SC35 nuclear domains of T. castaneum oocytes are possibly involved in mRNP but not snRNP biogenesis. PMID:23686847

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

2013-10-01

118

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

PubMed

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

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

2009-12-01

119

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

PubMed Central

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.

Virant-Klun, Irma; Krijgsveld, Jeroen

2014-01-01

120

An intercellular pathway for glucose transport into mouse oocytes  

PubMed Central

Glucose is an essential nutrient for mammalian cells. Emerging evidence suggests that glucose within the oocyte regulates meiotic maturation. However, it remains controversial as to whether, and if so how, glucose enters oocytes within cumulus-oocyte complexes (COCs). We used a fluorescent glucose derivative (6-NBDG) to trace glucose transport within live mouse COCs and employed inhibitors of glucose transporters (GLUTs) and gap junction proteins to examine their distinct roles in glucose uptake by cumulus cells and the oocyte. We showed that fluorescent glucose enters both cumulus-enclosed and denuded oocytes. Treating COCs with GLUT inhibitors leads to simultaneous decreases in glucose uptake in cumulus cells and the surrounded oocyte but no effect on denuded oocytes. Pharmacological blockade of of gap junctions between the oocyte and cumulus cells significantly inhibited fluorescent glucose transport to oocytes. Moreover, we find that both in vivo hyperglycemic environment and in vitro high-glucose culture increase free glucose levels in oocytes via gap junctional channels. These findings reveal an intercellular pathway for glucose transport into oocytes: glucose is taken up by cumulus cells via the GLUT system and then transferred into the oocyte through gap junctions. This intercellular pathway may partly mediate the effects of high-glucose condition on oocyte quality.

Wang, Qiang; Chi, Maggie M.; Schedl, Tim

2012-01-01

121

Apoptosis Maintains Oocyte Quality in Aging Caenorhabditis elegans Females  

PubMed Central

In women, oocytes arrest development at the end of prophase of meiosis I and remain quiescent for years. Over time, the quality and quantity of these oocytes decreases, resulting in fewer pregnancies and an increased occurrence of birth defects. We used the nematode Caenorhabditis elegans to study how oocyte quality is regulated during aging. To assay quality, we determine the fraction of oocytes that produce viable eggs after fertilization. Our results show that oocyte quality declines in aging nematodes, as in humans. This decline affects oocytes arrested in late prophase, waiting for a signal to mature, and also oocytes that develop later in life. Furthermore, mutations that block all cell deaths result in a severe, early decline in oocyte quality, and this effect increases with age. However, mutations that block only somatic cell deaths or DNA-damageinduced deaths do not lower oocyte quality. Two lines of evidence imply that most developmentally programmed germ cell deaths promote the proper allocation of resources among oocytes, rather than eliminate oocytes with damaged chromosomes. First, oocyte quality is lowered by mutations that do not prevent germ cell deaths but do block the engulfment and recycling of cell corpses. Second, the decrease in quality caused by apoptosis mutants is mirrored by a decrease in the size of many mature oocytes. We conclude that competition for resources is a serious problem in aging germ lines, and that apoptosis helps alleviate this problem.

Andux, Sara; Ellis, Ronald E.

2008-01-01

122

Baccharis pteronioides toxicity in livestock and hamsters.  

PubMed

Baccharis pteronioides DC has been intermittently associated with livestock poisoning in the southwestern United States. In 2004, nearly 100 cows were reported poisoned by B. pteronioides in southern New Mexico. Initial field studies and postmortem examinations found drought conditions, evidence of B. pteronioides consumption, and a reported mortality of nearly 40%. Because postmortem materials were unsuitable for further examination, plant samples were collected for feeding trials and chemical evaluation. Forty-eight Syrian hamsters (8 weeks old) were randomly divided into 4 groups and dosed with 0, 50, 100, and 200 mg of B. pteronioides for 10 days. After dosing, the hamsters were necropsied; sera were analyzed biochemically; and tissues were collected and evaluated histologically. The hamsters treated with 200 mg and several of the 100-mg animals developed anorexia and diarrhea. These animals developed multiple hemorrhagic infarcts in the liver and kidney, with severe hemorrhagic enteritis. Histologically, the higher-dosed animals had severe necrotizing vasculitis with vascular thrombosis of hepatic and renal vessels. Many glomerular capillaries contained fibrin thrombi. The superficial intestinal and colonic mucosa was necrotic, with extensive hemorrhage and proliferation of luminal bacteria. Lower-dosed animals had mild hepatocellular swelling, with proliferation of intestinal and gastric bacteria and yeast. The findings indicate that at high doses, B. pteronioides is toxic to hamsters and produces lesions that are very similar to bacterial endotoxin-produced vasculitis and infarction. Research to purify and identify the toxin, the toxic dose, and mechanism of toxicity are ongoing. PMID:19286499

Stegelmeier, Bryan L; Sani, Yulvian; Pfister, James A

2009-03-01

123

Experimental histoplasmosis in temporarily hibernating hamsters  

Microsoft Academic Search

Summary Although occasional hibernation was observed in golden hamsters kept at low temperatures during the winter months, the periods of hibernation were apparently too short to induce conversion of the yeast cells ofH. capsulatum into the mycelial phase or to prevent conversion into the yeast phase when inoculated in the mycelial phase.

Karlhanns Salfelder; Krishan K. Sethi; Jan Schwarz

1965-01-01

124

Overview: Hard Rock Penetration  

Microsoft Academic Search

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

Dunn

1992-01-01

125

Overview - Hard Rock Penetration  

Microsoft Academic Search

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

Dunn; James C

1992-01-01

126

Genome analyses of single human oocytes.  

PubMed

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

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

127

Capacitative calcium entry mechanism in porcine oocytes.  

PubMed

The presence of the capacitative Ca(2+) entry mechanism was investigated in porcine oocytes. In vitro-matured oocytes were treated with thapsigargin in Ca(2+)-free medium for 3 h to deplete intracellular calcium stores. After restoring extracellular calcium, a large calcium influx was measured by using the calcium indicator dye fura-2, indicating capacitative Ca(2+) entry. A similar divalent cation influx could also be detected with the Mn(2+)-quench technique after inositol 1,4,5-triphosphate-induced Ca(2+) release. In both cases, lanthanum, the Ca(2+) permeable channel inhibitor, completely blocked the influx caused by store depletion. Heterologous expression of Drosophila trp in porcine oocytes enhanced the thapsigargin-induced Ca(2+) influx. Polymerase chain reaction cloning using primers that were designed based on mouse and human trp sequences revealed that porcine oocytes contain a trp homologue. As in other cell types, the capacitative Ca(2+) entry mechanism might help in refilling the intracellular stores after the release of Ca(2+) from the stores. Further investigation is needed to determine whether the trp channel serves as the capacitative Ca(2+) entry pathway in porcine oocytes or is simply activated by the endogenous capacitative Ca(2+) entry mechanism and thus contributes to Ca(2+) influx. PMID:11870073

Machty, Zoltn; Ramsoondar, Jagdeece J; Bonk, Aaron J; Bondioli, Kenneth R; Prather, Randall S

2002-03-01

128

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...

129

Proteasomal interference prevents zona pellucida penetration and fertilization in mammals.  

PubMed

The ubiquitin-proteasome pathway has been implicated in the penetration of ascidian vitelline envelope by the fertilizing spermatozoon (Sawada et al., Proc Natl Acad Sci U S A 2002; 99:1223-1228). The present study provides experimental evidence demonstrating proteasome involvement in the penetration of mammalian zona pellucida (ZP). Using porcine in vitro fertilization as a model, penetration of ZP was completely inhibited by specific proteasomal inhibitors MG-132 and lactacystin. Three commercial rabbit sera recognizing 20S proteasomal core subunits beta-1i, beta-2i, alpha-6, and beta-5 completely blocked fertilization at a very low concentration (i.e., diluted 1/2000 to 1/8000 in fertilization medium). Neither proteasome inhibitors nor antibodies had any effects on sperm-ZP binding and acrosome exocytosis in zona-enclosed oocytes or on fertilization rates in zona-free oocytes, which were highly polyspermic. Consistent with a possible role of ubiquitin-proteasome pathway in ZP penetration, ubiquitin and various alpha and beta type proteasomal subunits were detected in boar sperm acrosome by specific antibodies, immunoprecipitated and microsequenced by MALDI-TOF from boar sperm extracts. Antiubiquitin-immunoreactive substrates were detected on the outer face of ZP by epifluorescence microscopy. This study therefore provides strong evidence implicating the ubiquitin-proteasome pathway in mammalian fertilization and zona penetration. This finding opens a new line of acrosome/ZP research because further studies of the sperm acrosomal proteasome can provide new tools for the management of polyspermia during in vitro fertilization and identify new targets for contraceptive development. PMID:15253927

Sutovsky, Peter; Manandhar, Gaurishankar; McCauley, Tod C; Caamao, Jose Nestor; Sutovsky, Miriam; Thompson, Winston E; Day, Billy N

2004-11-01

130

Cdc6 synthesis regulates replication competence in Xenopus oocytes  

Microsoft Academic Search

The early division cycles of an embryo rely on the oocyte's ability to replicate DNA. During meiosis, oocytes temporarily lose this ability. After a single round of pre-meiotic S-phase, oocytes enter meiosis and rapidly arrest at prophase of meiosis I (G2). Upon hormonal stimulation, arrested oocytes resume meiosis, re-establish DNA replication competence in meiosis I shortly after germinal vesicle breakdown

Elizabeth Whitmire; Bettina Khan; Martine Cou

2002-01-01

131

Cellular and molecular mechanisms of various types of oocyte aging  

Microsoft Academic Search

It is well established that age-related decline of a woman's fertility is related to the poor developmental potential of her\\u000a gametes. The age-associated decline in female fertility is largely attributable to the oocyte aging caused by ovarian aging.\\u000a Age-associated oocyte aging results in a decrease in oocyte quality. In contrast to ovarian aging, there is a concept of postovulatory\\u000a oocyte

Toshifumi Takahashi; Hideki Igarashi; Mitsuyoshi Amita; Shuichiro Hara; Hirohisa Kurachi

132

[Consequences of stimulation on oocyte quality].  

PubMed

In all IVF programs, ovarian stimulation may lead to a hyperstimulation syndrome. Mild ovarian stimulations are suggested to reduce this complication of infertility treatment. In this article, we wonder what the consequences of different stimulations are on oocytes quality. The quality of the oocyte being the major factor of the development of a good embryo, it can be evaluated by direct or indirect techniques. Some are subjective. Several studies seem to show that ovarian stimulation has an influence on oocye quality, especially at a chromosomic and perharps, epigenetic level. It seems that a mild stimulation (for example with GnRH antagonist) selects less oocytes but with a better quality and produces euploid embryos. The uterine transfer of this kind of embryo should lead to an ongoing pregnancy. PMID:17822936

Clment, P

2007-09-01

133

Portrait of an oocyte: our obscure origin  

PubMed Central

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.

Gosden, Roger; Lee, Bora

2010-01-01

134

LINE-1 of evidence for fetal oocyte attrition by retrotransposon.  

PubMed

Fetal oocytes in mammals undergo extensive apoptosis during development. In this issue of Developmental Cell, Malki et al. (2014) provide insight into how and why such massive oocyte loss occurs through the demonstration that the expression level of LINE-1 retrotransposon defines the survival threshold and thus viability of fetal oocytes. PMID:24914555

Chuma, Shinichiro

2014-06-01

135

The human cumulus-oocyte complex gene expression profile  

Microsoft Academic Search

BACKGROUND: The understanding of the mechanisms regulating human oocyte maturation is still rudimentary. We have identified transcripts differentially expressed between immature and mature oocytes and cumulus cells. METHODS: Using oligonucleotide microarrays, genome-wide gene expression was studied in pooled immature and mature oocytes or cumulus cells from patients who underwent IVF. RESULTS: In addition to known genes, such as DAZL, BMP15

Said Assou; Tal Anahory; Vronique Pantesco; Tanguy Le Carrour; Franck Pellestor; Bernard Klein; Lionel Reyftmann; Herv Dechaud; John De Vos; Samir Hamamah

136

Oocyte markets: women's reproductive work in embryonic stem cell research  

Microsoft Academic Search

Somatic cell nuclear transfer (SCNT) research, otherwise known as therapeutic cloning, requires large numbers of research oocytes, placing pressure on an already limited supply. In the UK, Canada, Australia, Singapore and most of Western Europe, oocytes are made available through modestly reimbursed donation, and, owing to the onerous nature of donation, the existing demand for reproductive oocytes far outstrips availability.

Catherine Waldby

2008-01-01

137

Cryopreservation of supernumerary oocytes in IVF\\/ICSI cycles  

Microsoft Academic Search

BACKGROUND: The aim of the present study is to investigate cryopreservation of oocytes in patients refusing embryo cryopreservation for ethical reasons, patients from whom no sperm could be retrieved and patients with enough oocytes to yield a number of fresh and cryopreserved embryos to transfer. METHODS: A total of 2900 oocytes out of 6216 retrieved were cryopreserved in 286 patients

P. E. Levi Setti; E. Albani; P. V. Novara; A. Cesana; G. Morreale

2005-01-01

138

Evaluation of Microencapsulated Penetrant Inspection.  

National Technical Information Service (NTIS)

Presented are the results of a program to evaluate the merits and short-comings of microencapsulated penetrants for use in fluorescent penetrant inspection to evaluate capsule and spray parameters to effectively use microencapsulated penetrants, and to co...

J. M. Portaz

1980-01-01

139

Microencapsulated Fluorescent Dye Penetrant.  

National Technical Information Service (NTIS)

Microencapsulated fluorescent dye pentrant materials were evaluated for feasibility as a technique to detect cracks on metal surfaces when applied as a free flowing dry powder. Various flourescent dye solutions in addition to a commercial penetrant (Zyglo...

S. Allinikov

1979-01-01

140

Bioactivation of diethylstilbestrol by the Syrian hamster kidney  

SciTech Connect

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%.

Adams, S.P.

1987-01-01

141

Campylobacter cinaedi is normal intestinal flora in hamsters.  

PubMed

During the course of studies to reproduce proliferative enteritis in hamsters, Campylobacter cinaedi was recovered from the feces of the majority of healthy hamsters obtained from two commercial sources. The organisms were cultured by using filtration, a nonselective medium, and a microaerophilic atmosphere containing hydrogen. Isolation was hindered by the fastidious nature of C. cinaedi and by the presence of other Campylobacter species in the hamster intestine. All hamster C. cinaedi isolates were phenotypically similar to C. cinaedi ATCC 35683. Comparison of whole-cell protein profiles of one hamster isolate with a reference strain of C. cinaedi by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting with C. cinaedi-specific rabbit antiserum supported the phenotypic identification of these isolates. Hamsters may be an animal reservoir for human C. cinaedi infections. PMID:2768458

Gebhart, C J; Fennell, C L; Murtaugh, M P; Stamm, W E

1989-07-01

142

Sperm and Oocyte Communication Mechanisms Controlling C. elegans Fertility  

PubMed Central

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.

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

2010-01-01

143

Increased expression of ERp57 in rat oocytes during meiotic maturation is associated with sperm-egg fusion.  

PubMed

Oocyte meiotic maturation is a developmental transition that starts during germinal-vesicle breakdown and ends at the arrest in metaphase of meiosis II. This transition is associated with changes to both the proteins that are synthesized and the abundance/distribution of post-translational modifications that are crucial for subsequent fertilization and embryogenesis. Here, we isolated and cultured rat oocytes in vitro during both metaphase of meiosis I (MI) and meiosis II (MII) stages, respectively, and then compared their proteomic profiles by high-resolution, two-dimensional gel electrophoresis (2DE) followed by mass spectrometry. We found that the expression of five proteins was up-regulated while six proteins were down-regulated when comparing MI to MII oocytes. The expression of ERp57, an endoplasmic reticulum chaperone, underwent a dramatic increase between MI and MII oocytes, and became concentrated in a dome-shaped area of the cell surface within the microvillar region. A similar profile was observed during spermatogenesis, and sperm ERp57 eventually localized to the head and flagellum surfaces, finally ending in the equatorial region of acrosome-reacted sperm. Given the localization pattern, we tested and found that a polyclonal antiserum created against recombinant rat ERp57 significantly inhibited spermatozoa from penetrating zona pellucida-free oocytes without affecting either sperm motility or the acrosome reaction. These results indicate that ERp57 expression on oocytes, and possibly sperm, plays an important physiological role during sperm-egg fusion. Mol. Reprod. Dev. 81: 315-325, 2014. 2014 Wiley Periodicals, Inc. PMID:24415168

Liu, Yue; Zhu, Yemin; Wu, Xiaohui; Li, Yandong; Guo, Qiangsu; Li, Weiping; Ding, Zhide

2014-04-01

144

Effect of an environmentally relevant metabolized organochlorine mixture on porcine cumulus-oocyte complexes.  

PubMed

Organochlorine compounds and their metabolites bioaccumulate and have been detected in follicular and genital tract fluids of humans and animals. This study was designed to investigate the effect of a metabolised organochlorine mixture, extracted from plasma of sows treated with an environmentally relevant organochlorine mixture in the course of a previous study, on porcine cumulus-oocyte complexes (COCs) in vitro. The major component of the metabolised mixture is 2,2-bis(p-chlorophenyl)-1,1-dichloroethylene (p,p'-DDE) at 15.1 mg/l, which accounts for 40.7% of the total extract. Polychlorinated biphenyls (PCBs) account for 30.8% of the extract and hydroxylated PCB metabolites (OH-PCBs) for 11.8%. Exposure of COCs to the metabolised mixture induced a decrease of apoptotic cumulus cells at low concentrations and an increase at higher concentrations following a U-shaped curve (p=0.0106), with the intermediate treatment (3.6 microg/l OH-PCBs) significantly reducing apoptosis compared to the extraction control (p=0.05). However, the metabolised mixture did not affect cumulus expansion, oocyte maturation, penetration, development to blastocyst, or the number of cells per blastocyst. This study also indicates that organochlorine metabolites similar in concentrations to levels found in Arctic populations can affect growing cumulus-oocyte complexes without inducing an overt toxicological response. PMID:17158027

Campagna, Cline; Ayotte, Pierre; Sirard, Marc-Andr; Arsenault, Guylne; Laforest, Jean-Paul; Bailey, Janice L

2007-02-01

145

Effects of ??tetrahydrocannabinol on hamster fetuses  

Microsoft Academic Search

Pregnant golden Syrian hamsters were given single or multiple intragastric doses of ??tetrahydrocannabinol (??THC) dissolved in olive oil. Dose levels of 125, 250, or 500 mg\\/kg were administered by gavage once during gestational days 712 and of 25, 50, or 100 mg\\/kg daily on days 710, 912, or 712 in the two experimental series. Untreated and vehicle?injected controls were used

M. G. Joneja

1977-01-01

146

Oncogenity of BK virus for immunosuppressed hamsters  

Microsoft Academic Search

Summary Tumors were induced by BK virus (BKV) inoculated intravenously in 3-week-old Syrian golden hamsters immunosuppressed with anti-lymphocyte serum or methylprednisolone acetate alone or in association with ?-radiation (60Co). The induced neoplasms were ependymoma, carcinoma of pancreatic islets, lymphoma, osteosarcoma, undifferentiated sarcoma, kidney and renal pelvis carcinoma, pheochromocytoma and hemangiosarcoma. High levels of insulin and glucagon and altered concentrations of

A. Corallini; G. Altavilla; L. Carra; M. P. Grossi; G. Federspil; A. Caputo; M. Negrini; G. Barbanti-Brodano

1982-01-01

147

Mouse Oocyte Killing by Neutrons: Target Considerations.  

National Technical Information Service (NTIS)

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 (as...

T. Straume R. L. Dobson

1985-01-01

148

Age-Dependent Radiosensitivity of Mouse Oocytes.  

National Technical Information Service (NTIS)

It has been shown that there are three distinct phases of radiosensitivity in oocytes of prepubertal mice: a period of rapidly increasing sensitivity between 0 and 4 days of age; a period of consistent, high sensitivity between 5 and 18 days of age; and a...

C. Koehler

1976-01-01

149

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

PubMed

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

Tesarik, J; Sousa, M; Mendoza, C

1995-06-01

150

Selection of Ovine Oocytes by Brilliant Cresyl Blue Staining  

PubMed Central

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.

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

2012-01-01

151

Somatic cell-oocyte interactions in mouse oogenesis: stage-specific regulation of mouse oocyte protein phosphorylation by granulosa cells.  

PubMed

The relative rate of synthesis of a number of proteins and the protein phosphorylation pattern of growing and fully grown oocytes were influenced by the presence of granulosa cells. In particular, a 74-kDa phosphorylated protein was detected only in granulosa cell-enclosed growing mouse oocytes. When reaggregated with granulosa cells, the growing oocyte displayed the phosphorylated form of the 74-kDa protein but when oocytes were cultured on Sertoli cell monolayers or in granulosa cell-conditioned medium the 74-kDa protein was not phosphorylated. We propose that (1) granulosa cells regulate protein phosphorylation in mouse oocytes; (2) a 74-kDa protein is phosphorylated only in growing oocytes when surrounded by granulosa cells; and (3) granulosa cells, but not Sertoli cells, are competent to send the appropriate "signal" to the growing oocyte. PMID:2707483

Colonna, R; Cecconi, S; Tatone, C; Mangia, F; Buccione, R

1989-05-01

152

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

PubMed

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

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

2013-12-01

153

Age-associated alteration of oocyte-specific gene expression in polar bodies potential markers of oocyte competence  

PubMed Central

Objective To confirm that oocyte-specific mRNAs are detectable in the polar body (PB) of MII oocytes and determine the effect of age on oocyte-specific transcript levels. Design Prospective study Setting Hospital-based academic research laboratory Animals CD1 female mice Intervention(s) Aged (4050 weeks) and young (79 weeks) mice were administered pregnant mare's serum gonadotropin (PMSG) and human chorionic gonadotrophin (hCG). Oocytes were fertilized in vitro to assess fertilization and developmental competence. MII oocytes were obtained and first PBs was removed. mRNA from each PB and its sibling oocyte was reverse transcribed and analyzed by real-time quantitative PCR. Main Outcome Measure(s) Fertilization and developmental rates and expression of 6 oocyte-specific genes (Bmp15, Gdf9, H1foo, Nlrp5, Tcl1, and Zp3) in PBs and sibling oocytes from young vs. aged mice. Result(s) Oocytes from aged mice had lower developmental competence. Four genes (H1foo, Nlrp5, Tcl1, and Zp3) were differentially expressed in aged vs. young oocytes (P < 0.05). All 6 transcripts were present in PBs from aged and young mice at lower levels than in the sibling oocytes; transcript levels were lower in aged PBs compared with young PBs (P < 0.05). Conclusion(s) There is a significant difference in the transcript levels of oocyte-specific genes in aged vs. young PB that correlates with age-related decreases in oocyte competence. Differences in gene expression in PB may be potential biomarkers of MII oocyte competence.

Jiao, Ze-Xu; Xu, Min; Woodruff, Teresa K

2012-01-01

154

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

PubMed Central

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)

Yoshida, S; Plant, S

1991-01-01

155

The human cumulus--oocyte complex gene-expression profile  

PubMed Central

BACKGROUND The understanding of the mechanisms regulating human oocyte maturation is still rudimentary. We have identified transcripts differentially expressed between immature and mature oocytes, and cumulus cells. METHODS Using oligonucleotides microarrays, genome wide gene expression was studied in pooled immature and mature oocytes or cumulus cells from patients who underwent IVF. RESULTS In addition to known genes such as DAZL, BMP15 or GDF9, oocytes upregulated 1514 genes. We show that PTTG3 and AURKC are respectively the securin and the Aurora kinase preferentially expressed during oocyte meiosis. Strikingly, oocytes overexpressed previously unreported growth factors such as TNFSF13/APRIL, FGF9, FGF14, and IL4, and transcription factors including OTX2, SOX15 and SOX30. Conversely, cumulus cells, in addition to known genes such as LHCGR or BMPR2, overexpressed cell-tocell signaling genes including TNFSF11/RANKL, numerous complement components, semaphorins (SEMA3A, SEMA6A, SEMA6D) and CD genes such as CD200. We also identified 52 genes progressively increasing during oocyte maturation, comprising CDC25A and SOCS7. CONCLUSION The identification of genes up and down regulated during oocyte maturation greatly improves our understanding of oocyte biology and will provide new markers that signal viable and competent oocytes. Furthermore, genes found expressed in cumulus cells are potential markers of granulosa cell tumors.

Assou, Said; Anahory, Tal; Pantesco, Veronique; Le Carrour, Tanguy; Pellestor, Franck; Klein, Bernard; Reyftmann, Lionel; Dechaud, Herve; De Vos, John; Hamamah, Samir

2006-01-01

156

Proteome of mouse oocytes at different developmental stages  

PubMed Central

The mammalian oocyte possesses powerful reprogramming factors, which can reprogram terminally differentiated germ cells (sperm) or somatic cells within a few cell cycles. Although it has been suggested that use of oocyte-derived transcripts may enhance the generation of induced pluripotent stem cells, the reprogramming factors in oocytes are undetermined, and even the identified proteins composition of oocytes is very limited. In the present study, 7,000 mouse oocytes at different developmental stages, including the germinal vesicle stage, the metaphase II (MII) stage, and the fertilized oocytes (zygotes), were collected. We successfully identified 2,781 proteins present in germinal vesicle oocytes, 2,973 proteins in MII oocytes, and 2,082 proteins in zygotes through semiquantitative MS analysis. Furthermore, the results of the bioinformatics analysis indicated that different protein compositions are correlated with oocyte characteristics at different developmental stages. For example, specific transcription factors and chromatin remodeling factors are more abundant in MII oocytes, which may be crucial for the epigenetic reprogramming of sperm or somatic nuclei. These results provided important knowledge to better understand the molecular mechanisms in early development and may improve the generation of induced pluripotent stem cells.

Wang, Shufang; Kou, Zhaohui; Jing, Zhiyi; Zhang, Yu; Guo, Xinzheng; Dong, Mengqiu; Wilmut, Ian; Gao, Shaorong

2010-01-01

157

Developmental potential of in vitro or in vivo matured oocytes.  

PubMed

Summary This study compared the embryological features of mature and immature oocytes (different stages) collected from stimulated cycles of in vitro fertilization (IVF). Immature oocytes were identified, classified as PI (prophase I - germinal vesicle, GV) or MI (metaphase I), were matured in vitro and fertilized using the intra-cytoplasmic sperm injection (ICSI) technique. Fertilization potential, cleavage, and subsequent transfer/cryopreservation of the embryos derived from these in vitro matured oocytes were compared with those of in vivo matured oocytes (collected at the MII stage). The characteristics of embryos derived from gametes recovered in the MI and MII stages were similar. The fertilization rate of immature oocytes recovered in PI was significantly lower than that of MII oocytes (P = 0.031), and the cleavage rate of the PI group was also lower than that of the MI (P = 0.004) and MII (P < 0.001) groups. In vitro maturation of MI oocytes is a suitable alternative when immature oocytes are recovered, as their characteristics and development are similar to those of in vivo matured oocytes. Optimization of outcomes for PI oocytes will require development of techniques that can distinguish which of these gametes will mature and fertilize. PMID:23735140

Alcoba, Diego D; Pimentel, Anita M; Brum, Ilma S; Corleta, Helena E

2013-06-01

158

Maternal restraint stress diminishes the developmental potential of oocytes.  

PubMed

Although studies of both humans and animals suggest detrimental effects of psychological (restraint) stress on reproduction, reports concerning the direct effect of psychological (restraint) stress on the oocyte are few and conflicting. In the present study, a restraint system that allows mice free intake of feed and water while restraining their movement was established, and effects of maternal restraint on oocyte competence were examined by observing embryo development in vitro and in vivo. The results indicated that restraint stress applied to both gonadotropin-stimulated and unstimulated females during oocyte growth and maturation increased their plasma cortisol level but impaired ovulation and oocyte developmental potential. Injection of cortisol also decreased oocyte developmental potential in both stimulated and unstimulated mice. However, whereas restraint stress reduced the plasma follicle-stimulating hormone (FSH) level of unstimulated mice, injection of cortisol did not. Because the stimulated mice had received very high doses of FSH and luteinizing hormone from injection with equine chorionic gonadotropin injection, the results suggested that whereas cortisol acts directly on the ovary to damage the oocyte, restraint stress impairs oocyte competence by actions on both the hypothalamic-pituitary-gonadal and the hypothalamic-pituitary-adrenal axes. However, exposing the cumulus-oocyte complexes (COCs) to physiological levels of cortisol did not affect oocyte nuclear and cytoplasmic maturation in vitro. Thus, cortisol might have impaired ovulation and oocyte potential by an indirect effect on ovarian tissues other than the COCs. PMID:21123817

Zhang, Si-Yu; Wang, Jun-Zuo; Li, Jing-Jing; Wei, De-Li; Sui, Hong-Shu; Zhang, Zhen-Hua; Zhou, Ping; Tan, Jing-He

2011-04-01

159

Follicle environment and quality of in vitro matured oocytes.  

PubMed

In mammalian reproduction, the oocyte depends on the ovarian follicle for most of its growth. They form a bipolar partnership and the status of one will impact the functioning of the other. When oocytes are removed from their follicle by ovulation, they have normally completed all the steps required to begin their journey into the oviduct and drive the early embryonic development. When oocytes are removed from their follicle before natural ovulation, the process by which they acquire all the important components for their journey might not be completed and their ability to mature, fertilize or develop into embryos or to term might be compromised. Animal models have been useful to define the important steps required for the oocyte's growth phase, and in the mouse, when the oocyte has reached its full size, the program is ready. This is not the case in larger mammals where the completion of growth does not ensure that the oocyte is fully capable of undergoing all the steps to the embryo and to term. The final steps of oocyte preparation also involve a progressive condensation of the chromatin that may facilitate normal maturation but may also indirectly reduce the lifespan of the oocyte. In such a scenario, the oocyte would have an expiration date when fully competent. In humans, a number of indications may justify the aspiration of oocytes from unstimulated patients and the development of an in vitro maturation (IVM) process that would allow fertilization and subsequent development. This objective could be realized by a better understanding of the essential follicular contribution required before removing the oocyte. Therefore, this review will focus on the large animal models where IVM has been used and studied for more than 25years. The status of the follicle at the time of oocyte recovery and the status of the oocyte's chromatin will be described in detail as they have a significant impact on the outcome. PMID:21394521

Sirard, Marc-Andr

2011-06-01

160

Autophagic activation in vitrified-warmed mouse oocytes.  

PubMed

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

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

2014-07-01

161

Development of primordial follicles in the hamster: role of estradiol-17beta.  

PubMed

The role of E2 on primordial follicle formation was examined by treating neonatal hamsters with 1 or 2 microg estradiol cypionate (ECP) at age postnatal d 1 (P1) and P4 or by in vitro culture of embryonic d 15 (E15) ovaries with 1, 5, or 10 ng/ml estradiol-17beta (E2). The specificity of E2 action was examined by ICI 182,780. One microgram of ECP maintained serum levels of E2 within the physiological range, significantly reduced apoptosis, and stimulated the formation and development of primordial follicles. In contrast, 2 microg ECP increased serum E2 levels to 400 pg/ml and had significantly less influence on primordial follicle formation. In vivo, ICI 182,780 significantly increased apoptosis and caused a modest reduction in primordial follicle formation. The formation and development of primordial follicles in vitro increased markedly with 1 ng/ml E2, and the effect was blocked by ICI 182,780. Higher doses of E2 had no effect on primordial follicle formation but significantly up-regulated apoptosis, which was blocked by ICI 182,780. CYP19A1 mRNA expression occurred by E13 and increased with the formation of primordial follicles. P4 ovaries synthesized E2 from testosterone, which increased further by FSH. Both testosterone and FSH maintained ovarian CYP19A1 mRNA, but FSH up-regulated the expression. These results suggest that neonatal hamster ovaries produce E2 under FSH control and that E2 action is essential for the survival and differentiation of somatic cells and the oocytes leading to the formation and development of primordial follicles. This supportive action of E2 is lost when hormone levels increase above a threshold. PMID:17194746

Wang, Cheng; Roy, Shyamal K

2007-04-01

162

Cryotop vitrification of human oocytes results in high survival rate and healthy deliveries  

Microsoft Academic Search

Vitrification, an ultra-rapid cooling technique, offers a new perspective in attempts to develop an optimal cryopreservation procedure for human oocytes and embryos. To further evaluate this method for human oocytes, 796 mature oocytes (metaphase II) were collected from 120 volunteers. Since Italian legislation allows the fertilization of a maximum of only three oocytes per woman, there were 463 supernumerary oocytes;

Monica Antinori; Emanuele Licata; Gianluca Dani; Fabrizio Cerusico; Caterina Versaci; Severino Antinori

2007-01-01

163

Circadian Organization oftau Mutant Hamsters: Aftereffects and Splitting  

Microsoft Academic Search

Homozygous tau mutant (?ss) hamsters show an extremely short (20 h) circadian period (?) that is attributable to altered enzymatic activity of casein kinase 1?. It has been proposed that coupling of constituent circadian oscillators is strengthened in ?ss hamsters, explaining their tendency to show strong resetting after prolonged exposure to constant darkness. To evaluate further the circadian organization of

Eric L. Bittman; Mary K. Costello; Judy McKinley Brewer

2007-01-01

164

Open versus closed oocyte vitrification system: a prospective randomized sibling-oocyte study.  

PubMed

Vitrification has been successfully applied in the cryopreservation of oocytes and embryos. It can be achieved either by direct (open system) or indirect (closed system) contact with liquid nitrogen. Unlike embryo vitrification, few reports have been published regarding oocyte vitrification in closed systems. In order to validate the effectiveness of a closed and aseptic vitrification approach for oocyte cryopreservation, a prospective, randomized study was performed. Sibling oocytes donated from the same donor were randomly and equally assigned into closed or open vitrification groups. A total of 75 vitrification-warming cycles were performed in each group. Apart from the survival rate (82.9% versus 91.0%, P<0.05), no statistically significant differences were observed in pregnancy (?-human chorionic gonadotrophin positive) (42.7% versus 33.3%), clinical pregnancy (36.0% versus 28.0%), implantation (13.8% versus 10.1%), ongoing pregnancy (33.3% versus 24.0%) and live birth (36.0% versus 24.0%) rates between the closed and open groups, and 27 and 18 healthy babies were born, respectively. This study shows that the replacement of the open vitrification system by a closed system has no impact on clinical pregnancy and implantation rates. Therefore, the closed vitrification system provides an aseptic alternative to the open method for oocyte vitrification. PMID:23602678

Papatheodorou, A; Vanderzwalmen, P; Panagiotidis, Y; Prapas, N; Zikopoulos, K; Georgiou, I; Prapas, Y

2013-06-01

165

A penetration mechanics database  

NASA Astrophysics Data System (ADS)

A penetration mechanics database has been compiled that contains experimental results from a variety of researchers of different nationalities (German, French, English, Canadian, and American), during different decades (the 1960's to the 1990's), and with different purposes or objectives (space debris impact, armor design and evaluation, penetrator design and evaluation, and theoretical verification). The data fall naturally into three subgroupings: (1) penetration into semi-infinite targets; (2) perforation of finite-thickness targets; and (3) penetration from multiple impact (segmented rods). Data collection was primarily limited to experiments conducted against generic type targets, and emphasis was placed on data that would be more relevant to heavy armor issues. This diverse collection of data for metallic targets has been tabulated; data tables include the initial impact conditions, the projectile and target geometries and materials, and the measured response. Information on the materials, e.g., hardness of yield strength, along with impact information such as yaw has been tabulated if available. Graphical displays of the data have been used to summarize the data, and cross-plotting that combines data from different sources has also been provided.

Anderson, Charles E., Jr.; Morris, Bruce L.; Littlefield, David L.

1992-01-01

166

Single wall penetration equations  

NASA Technical Reports Server (NTRS)

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.

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

1991-01-01

167

Hypervelocity impact penetration mechanics  

Microsoft Academic Search

Inert dense metal penetrators having a mass and geometry capable of missile delivery offer significant potential for countering underground facilities at depths of tens of meters in hard rock. The proliferation of such facilities among countries whose support for terrorism and potential possession of Weapons of Mass Destruction (WMD) constitutes threats to world peace and U.S. Security. The Defense Threat

C. McFarland; P. Papados; M. Giltrud

2008-01-01

168

Jet penetration in glass  

SciTech Connect

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.

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

1991-05-01

169

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

PubMed Central

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.

Choi, Jung Kyu; He, Xiaoming

2013-01-01

170

Immunosuppressive Agents in Intracellular Infection: Besnoitiosis in Hamsters  

PubMed Central

When tested for their activity in suppressing the acquisition of immunity during acute Besnoitia infection of hamsters, antilymphocyte serum (ALS), aminopterin, cyclophosphamide, cortisol, and whole-body irradiation were the most active agents and effectively blocked the development of immunity during 4 to 12 days of immunization. Actinomycin D and chlorambucil were moderately active, and nitrogen mustard, 6-mercaptopurine, and vinblastine exhibited slight immunosuppressive activity. Established immunity was especially labile to cortisol and cortisone administration with generalized Besnoitia relapse occurring consistently in all hamsters; this occurred infrequently during cyclophosphamide treatment. Focal relapse was seen in chronically irradiated and ALS-treated hamsters, but the location of the lesions differed. Irradiated hamsters had lesions in the lungs and brain, whereas ALS-treated hamsters showed splenic relapse. Acquisition of immunity was more sensitive to suppression than established immunity and did not necessarily parallel antibody development and vice versa, emphasizing the importance of cellular over humoral factors in immunity to this intracellular parasite.

Wilson, H. R.; Frenkel, J. K.

1971-01-01

171

Immunosuppressive agents in intracellular infection: besnoitiosis in hamsters.  

PubMed

When tested for their activity in suppressing the acquisition of immunity during acute Besnoitia infection of hamsters, antilymphocyte serum (ALS), aminopterin, cyclophosphamide, cortisol, and whole-body irradiation were the most active agents and effectively blocked the development of immunity during 4 to 12 days of immunization. Actinomycin D and chlorambucil were moderately active, and nitrogen mustard, 6-mercaptopurine, and vinblastine exhibited slight immunosuppressive activity. Established immunity was especially labile to cortisol and cortisone administration with generalized Besnoitia relapse occurring consistently in all hamsters; this occurred infrequently during cyclophosphamide treatment. Focal relapse was seen in chronically irradiated and ALS-treated hamsters, but the location of the lesions differed. Irradiated hamsters had lesions in the lungs and brain, whereas ALS-treated hamsters showed splenic relapse. Acquisition of immunity was more sensitive to suppression than established immunity and did not necessarily parallel antibody development and vice versa, emphasizing the importance of cellular over humoral factors in immunity to this intracellular parasite. PMID:16558050

Wilson, H R; Frenkel, J K

1971-06-01

172

Tumor-penetrating peptides.  

PubMed

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

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

2013-01-01

173

Tumor-Penetrating Peptides  

PubMed Central

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.

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

2013-01-01

174

Cryopreservation of Human Oocytes and Embryos  

Microsoft Academic Search

\\u000a With the advent of assisted reproductive technology, controled ovarian hyperstimulation (COH) is usually carried out to stimulate\\u000a the growth of multiple follicles and produce multiple oocytes. Accordingly, multiple embryos are transferred to the uterus\\u000a to increase the chances of success. However, multiple embryos can also increase the likelihood of multiple pregnancies, which\\u000a are accompanied by a whole series of complications

Barry Behr; Yimin Shu

175

In-vitro Maturation of Human Oocytes  

Microsoft Academic Search

\\u000a Since the birth of the first in-vitro fertilization (IVF) baby in 1978 [1], assisted reproductive technologies (ART) have\\u000a helped thousands of couples to have children through the use of ovarian stimulation [2]. IVF was first performed in a natural\\u000a cycle without any hormonal stimulation, and the oocyte retrieval was performed laparoscopically. However, natural cycle IVF\\u000a was soon replaced by ovarian

Ezgi Demirtas; Hananel Holzer; Weon-Young SON; Ri-Cheng Chain; Seang Lin Tan

176

Percutaneous penetration - methodological considerations.  

PubMed

Studies on percutaneous penetration are needed to assess the hazards after unintended occupational skin exposures to industrial products as well as the efficacy after intended consumer exposure to topically applied medicinal or cosmetic products. During recent decades, a number of methods have been developed to replace methods involving experimental animals. The results obtained from these methods are decided not only by the chemical or product tested, but to a significant degree also by the experimental set-up and decisions made by the investigator during the planning phase. The present MiniReview discusses some of the existing and well-known experimental in vitro and in vivo methods for studies of percutaneous penetration together with some more recent and promising methods. After this, some considerations and recommendations about advantages and limitations of the different methods and their relevance for the prediction of percutaneous penetration are given. Which method to prefer will depend on the product to be tested and the question asked. Regulatory guidelines exist for studies on percutaneous penetration, but researchers as well as regulatory bodies need to pay specific attention to the vehicles and solvents used in donor and sampling fluids so that it reflects in-use conditions as closely as possible. Based on available experimental data, mathematical models have been developed to aid predictions of skin penetration. The authors question the general use of the present mathematical models in hazard assessment, as they seem to ignore outliers among chemicals as well as the heterogeneity of skin barrier properties and skin conditions within the exposed populations. PMID:24373389

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

2014-07-01

177

Expression of Estrogen Receptor ? 36 (ESR36) in the Hamster Ovary throughout the Estrous Cycle: Effects of Gonadotropins  

PubMed Central

Estradiol-17? (E) plays an important role in ovarian follicular development. Evidence indicates that some of the effect of E is mediated by the transmembrane estrogen receptor. In this study, we examined the spatio-temporal expression of recently discovered ER?36 (ESR36), a splice variant of Esr1 and a receptor for non-genomic E signaling, in the hamster ovary during the estrous cycle and the role of gonadotropins and ovarian steroid hormones in ESR36 expression. ESR36 expression was high on estrus (D1?0900 h) and declined precipitously by proestrus (D4?0900 h) and remained low up to D4?1600 h. Immunofluorescence findings corroborated immunoblot findings and revealed that ESR36 was expressed only in the cell membrane of both follicular and non-follicular cells, except the oocytes. Ovarian ESR36 was capable of binding to the E-affinity matrix, and have different molecular weight than that of the ESR1 or GPER. Hypophysectomy (Hx) resulted in a marked decline in ESR36 protein levels. FSH and LH, alone or combined, markedly upregulated ESR36 protein in Hx hamsters to the levels observed in D1 hamsters, but neither E nor P had any effect. Inhibition of the gonadotropin surge by phenobarbital treatment on D4?1100 h attenuated ESR36 expression in D1?0900 h ovaries, but the decline was restored by either FSH or LH replacement on D4 afternoon. This is the first report to show that ESR36, which is distinct from ESR1 or GPER is expressed in the plasma membrane of ovarian follicular and non-follicular cells, binds to E and its expression is regulated directly by the gonadotropins. In light of our previous findings, the results suggest that ovarian cells contain at least two distinct membrane estrogen receptors, such as GPER and ESR36, and strongly suggest for a non-genomic action of E regulating ovarian follicular functions.

Chakraborty, Prabuddha; Roy, Shyamal K.

2013-01-01

178

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

PubMed

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

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

2011-12-01

179

Interference with the 19S proteasomal regulatory complex subunit PSMD4 on the sperm surface inhibits sperm-zona pellucida penetration during porcine fertilization  

Microsoft Academic Search

Proteolysis of ubiquitinated sperm and oocyte proteins by the 26S proteasome is necessary for the success of mammalian fertilization,\\u000a including but not limited to acrosomal exocytosis and sperm-zona pellucida (ZP) penetration. The present study examined the\\u000a role of PSMD4, an essential non-ATPase subunit of the proteasomal 19S regulatory complex responsible for proteasome-substrate\\u000a recognition, in sperm-ZP penetration during porcine fertilization in

Young-Joo Yi; Gaurishankar Manandhar; Miriam Sutovsky; Shawn W. Zimmerman; V?ra Jonkov; Fred W. van Leeuwen; Richard Oko; Chang-Sik Park; Peter Sutovsky

2010-01-01

180

Aging-related Changes in In Vitro-matured Bovine Oocytes: Oxidative Stress, Mitochondrial Activity and ATP Content After Nuclear Maturation.  

PubMed

The objective of this research was to clarify the aging-related changes in in vitro-matured bovine oocytes. Firstly, we examined the fertilization and embryonic development of bovine oocytes after 22 and 30-34 h of in vitro maturation (IVM). The oocytes after 30-34 h of IVM (penetrated by sperm at around 40 h after starting IVM) showed a lower developmental rate to blastocysts (P<0.01), although normal fertilization rates were similar regardless of IVM duration. In the next experiment, reactive oxygen species (ROS), mitochondrial activity and ATP content in oocytes after 20, 30 and 40 h of IVM were examined. The lowest level of ROS was found in the group subjected to 30 h of IVM. The mitochondrial activity and ATP content in the group subjected to 40 h of IVM were higher than in the group subjected to 20 h of IVM (P<0.01), and those in the group subjected to 30 h of IVM showed intermediate values. Thereafter, the mitochondrial activities at 3 days after in vitro fertilization in embryos derived from the oocytes subjected to 22 and 34 h of IVM were evaluated. In the group subjected to 34 h of IVM, high-polarized mitochondria were frequently observed at the periphery of blastomeres. The present results suggest that high mitochondrial activity observed in oocytes after prolonged IVM culture and localization of high-polarized mitochondria at the periphery of blastomeres during early embryonic development may be associated with the low developmental competence in aged bovine oocytes. PMID:24492658

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

2014-04-24

181

Fetuin inhibits zona pellucida hardening and conversion of ZP2 to ZP2f during spontaneous mouse oocyte maturation in vitro in the absence of serum.  

PubMed

The zona pellucida of mouse oocytes becomes resistant to chymotrypsin digestion, or "hardened", when spontaneous maturation occurs in serum-free medium (De Felici and Siracusa, Gam Res 1982; 6:107). The hardened zona pellucida is refractory to sperm penetration, thus preventing fertilization. Conversion of the zona pellucida glycoprotein ZP2 to ZP2f by a protease from precociously released oocyte cortical granules appears to be a major contributory factor of zona pellucida hardening (Ducibella et al., Dev Biol 1990; 137:46). Fetal bovine serum (FBS) prevents zona hardening and the ZP2 to ZP2f conversion during oocyte maturation in vitro (Downs et al., Gam Res 1986; 15:115; Ducibella et al., Dev Biol 1990; 137:46). This study was conducted to determine whether fetuin, a major glycoprotein constituent of FBS and a protease inhibitor, could prevent zona pellucida hardening during murine oocyte maturation in serum-free medium. Commercially available preparations of fetuin purified by three different methods were all active in inhibiting zona pellucida hardening in a concentration-dependent manner. Further chromatographic purification of one of these preparations indicated that the activity preventing zona pellucida hardening was associated specifically with fetuin. Fetuin also inhibited the conversion of ZP2 to ZP2f in a concentration-dependent manner during oocyte maturation in serum-free medium. Moreover, oocytes that matured in serum-free medium containing fetuin could be fertilized and could undergo preimplantation development to the blastocyst stage. These results indicate that fetuin, a component of FBS, inhibits zona pellucida hardening during oocyte maturation, and suggest that fetuin acts by preventing the proteolytic conversion of ZP2 to ZP2f by precociously released cortical granules. PMID:1705446

Schroeder, A C; Schultz, R M; Kopf, G S; Taylor, F R; Becker, R B; Eppig, J J

1990-11-01

182

Probing the mechanism of the hamster mitochondrial folate transporter by mutagenesis and homology modeling.  

PubMed

The mitochondrial folate transporter (MFT) was previously identified in human and hamster cells. Sequence homology of this protein with the inner mitochondrial membrane transporters suggested a domain structure in which the N- and C-termini of the protein are located on the mitochondrial intermembrane-facing surface, with six membrane-spanning regions interspersed by two intermembrane loops and three matrix-facing loops. We now report the functional significance of insertion of the c-myc epitope into the intermembrane loops and of a series of site-directed mutations at hamster MFT residues highly conserved in orthologues. Insertional mutagenesis in the first predicted intermembrane loop eliminated MFT function, but the introduction of a c-myc peptide into the second loop was without effect. Most of the hamster MFT residues studied by site-directed mutagenesis were remarkably resilient to these mutations, except for R249A and G192E, both of which eliminated folate transport activity. Homology modeling, using the crystal structure of the bovine ADP/ATP carrier (AAC) as a scaffold, suggested a similar three-dimensional structure for the MFT and the AAC. An ion-pair interaction in the AAC thought to be central to the mechanism of membrane penetration by ADP is predicted by this homology model to be replaced by a pi-cation interaction in MFT orthologues and probably also in other members of the family bearing the P(I/L)W motif. This model suggests that the MFT R249A and G192E mutations both modify the base of a basket-shaped structure that appears to constitute a trap door for the flux of folates into the mitochondrial matrix. PMID:17279620

Perchiniak, Erin; Lawrence, Scott A; Kasten, Shane; Woodard, B Ann; Taylor, Shirley M; Moran, Richard G

2007-02-13

183

Bidirectional communication between oocytes and follicle cells: ensuring oocyte developmental competence  

PubMed Central

Female fertility is determined to a large extent by the quality (developmental competence) of the oocyte as reflected in its ability to undergo meiosis, be fertilized, and give rise to a healthy embryo. Growth of the mammalian oocyte is coordinated with that of the follicle that encloses it by the actions of signals that pass in both directions between the germline and somatic components. This review summarizes what is known about the roles played by two different modes of intrafollicular signalling in oogenesis: paracrine factors activating receptors on the opposite cell type, and direct sharing of small molecules throughout the follicle via gap junction channels. Recent evidence indicates that these two modes of signalling interact to regulate oocyte growth and granulosa cell proliferation, and that defects in either can contribute to female infertility.

Kidder, Gerald M.; Vanderhyden, Barbara C.

2011-01-01

184

Cryopreservation of Mature Bovine Oocytes by Vitrification in Straws  

Microsoft Academic Search

Experiments were conducted to determine optimal conditions for vitrification ofin vitromatured bovine oocytes in straws. In the first series of experiments, the effects of stepwise addition before exposure of oocytes to vitrification solution consisting of 30% (v\\/v) ethylene glycol (EG) with 0.35 M sucrose was tested. The rates of morphological survival and cleavage of oocytes vitrified by the three-step addition

T. Otoi; K. Yamamoto; N. Koyama; S. Tachikawa; T. Suzuki

1998-01-01

185

Improved preparation of Xenopus oocytes for patch-clamp recording  

Microsoft Academic Search

We report a new technique for improving the efficiency of single-channel recording in the Xenopus oocyte expression system. Conventional methods of oocyte preparation (adequate for two-electrode voltage-clamp) often leave\\u000a residual adhesions between the vitelline and plasma membranes. As a result, removal of the vitelline membrane for patch-clamp\\u000a recording produces microscopic damage to the oocyte plasma membrane that interferes with the

H. Choe; H. Sackin

1997-01-01

186

Localization and regulation of ryanodine receptor in bovine oocytes.  

PubMed

We have previously reported that injection of ryanodine receptor agonists into mature bovine oocytes induces intracellular calcium release, indicative of the existence of ryanodine receptors. In this experiment, further evidence of the ryanodine receptor localization, and developmental regulation in bovine oocytes is presented. The possible physiological significance is also suggested. Using a rabbit antibody against the rabbit cardiac muscle ryanodine receptor, the ryanodine receptor was observed uniformly localized in the periphery of mature bovine oocytes, while a weak and discontinuous signal was observed in the germinal vesicle intact stage of bovine oocytes. As oocytes progress to the metaphase I stage, the ryanodine receptor localization became more intense and continuous, yet not comparable to that observed in the metaphase II oocytes. These modifications correlate with the intracellular calcium responsiveness to ryanodine. A 200-microM injection of ryanodine induces a low intracellular calcium transient in germinal vesicle-stage bovine oocytes, while peaked intracellular calcium transients are subsequently observed in the metaphase II-stage oocytes. However, no significant changes in the amplitude of intracellular calcium transients induced by 250 nM inositol 1,4,5-trisphosphate and 10 microM ionomycin were observed in oocytes at a comparable stage. Fertilization induced a significant decrease in ryanodine receptor signal; similar changes were also observed in oocytes injected with 200 microM ryanodine or incubated with 10 microM ionomycin. However, no changes in ryanodine signal were observed in oocytes injected with vehicle medium. Furthermore, injection of either ryanodine or inositol 1,4,5-trisphosphate induced subsequent pronuclear formation and cleavage. These data indicate that the ryanodine receptor is closely regulated and associated with early cellular changes following fertilization; stimulation of this receptor results in the activation of bovine oocytes, and it is likely that this receptor may play a role at fertilization. PMID:9475420

Yue, C; White, K L; Reed, W A; King, E

1998-02-01

187

Rapamycin Rescues the Poor Developmental Capacity of Aged Porcine Oocytes  

PubMed Central

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.

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

188

Oocyte-like cells induced from mouse spermatogonial stem cells  

PubMed Central

Background During normal development primordial germ cells (PGCs) derived from the epiblast are the precursors of spermatogonia and oogonia. In culture, PGCs can be induced to dedifferentiate to pluripotent embryonic germ (EG) cells in the presence of various growth factors. Several recent studies have now demonstrated that spermatogonial stem cells (SSCs) can also revert back to pluripotency as embryonic stem (ES)-like cells under certain culture conditions. However, the potential dedifferentiation of SSCs into PGCs or the potential generation of oocytes from SSCs has not been demonstrated before. Results We report that mouse male SSCs can be converted into oocyte-like cells in culture. These SSCs-derived oocytes (SSC-Oocs) were similar in size to normal mouse mature oocytes. They expressed oocyte-specific markers and gave rise to embryos through parthenogenesis. Interestingly, the Y- and X-linked testis-specific genes in these SSC-Oocs were significantly down-regulated or turned off, while oocyte-specific X-linked genes were activated. The gene expression profile appeared to switch to that of the oocyte across the X chromosome. Furthermore, these oocyte-like cells lost paternal imprinting but acquired maternal imprinting. Conclusions Our data demonstrate that SSCs might maintain the potential to be reprogrammed into oocytes with corresponding epigenetic reversals. This study provides not only further evidence for the remarkable plasticity of SSCs but also a potential system for dissecting molecular and epigenetic regulations in germ cell fate determination and imprinting establishment during gametogenesis.

2012-01-01

189

Comparative assessment of pre- and post-donation attitudes towards potential oocyte and embryo disposition and management among ovum donors in an oocyte donation programme  

Microsoft Academic Search

BACKGROUND: In anonymous oocyte donation programmes, the disposition of retrieved oocytes and subsequent embryo management are at the discretion of the IVF programme and the oocyte recipients, as donors waive all rights following their donation. Nonetheless, donors are routinely made aware of ways in which oocytes and resulting embryos may be used and elect to proceed with the process even

Julianne E. Zweifel; Meredith A. Rathert; Susan C. Klock; Heidi P. Walaski; Elizabeth A. Pritts; David L. Olive; Steven R. Lindheim

190

Susceptibility of bovine germinal vesicle-stage oocytes from antral follicles to direct effects of heat stress in vitro.  

PubMed

Delineation of maternal versus direct effects of heat stress in reducing development at the germinal vesicle (GV) stage is challenging, because oocytes spontaneously resume meiosis after removal from antral follicles. The use of S-roscovitine (inhibitor of p34(cdc2)/cyclin B kinase) to hold bovine oocytes at the GV stage without compromising early embryo development was previously validated in our laboratory. The objective of the present study was to assess the direct effects of an elevated temperature commonly seen in heat-stressed dairy cows on cumulus-oocyte complexes (COCs) held at the GV stage using 50 microM S-roscovitine. During roscovitine culture, GV-stage COCs (antral follicle diameter, 3-8 mm) were cultured at 38.5 or 41 degrees C. Thereafter, oocytes were removed from roscovitine medium and allowed to undergo in vitro maturation, fertilization, and culture. Zona pellucida hardening (solubility to 0.5% pronase), nuclear stage (Hoechst 33342), cortical granule type (lens culinaris agglutinin-fluorescein isothiocyanate [FITC]), and early embryo development were evaluated. Culture of GV-stage COCs at 41 degrees C increased the proportion that had type III cortical granules and reduced the proportion that progressed to metaphase II after in vitro maturation. Effects of 41 degrees C on zona pellucida hardening, fertilization (penetration, sperm per oocyte, pronuclear formation, and monospermic and putative embryos), and cleavage of putative zygotes were not noted. However, culture of GV-stage COCs at 41 degrees C for 6 h decreased the proportion of 8- to 16-cell embryos, whereas 41 degrees C for 12 h reduced blastocyst development. In summary, antral follicle COCs are susceptible to direct effects of elevated body temperature, which may account in part for reduced fertility in heat-stressed cows. PMID:15201201

Payton, Rebecca R; Romar, Raquel; Coy, Pilar; Saxton, Arnold M; Lawrence, Janelle L; Edwards, J Lannett

2004-10-01

191

Kinematics of trophectoderm projections and locomotion in the peri-implantation hamster blastocyst.  

PubMed

The behavior of golden hamster blastocysts was studied in vitro by continuous time-lapse videomicrography and computer imaging, during and immediately following escape from the zona pellucida. This study revealed numerous small cytoplasmic trophectoderm projections (TEPs) approximately 18 microns long that penetrated the zona pellucida both radically and tangentially and appeared to be actively involved in zona escape in vitro. After escape from their zonae, some blastocysts moved across the culture dish by an endogenous means of locomotion, most likely involving activity of the small TEPs. Several hours after zona escape, embryos expressed large TEPs up to 46 microns long that moved in an undulating manner and showed rapid cycles of extension and retraction; the timing of their appearance suggested that these TEPs are normally involved in attachment to the uterine epithelium. Embryos fixed in utero, during the developmental interval between zona loss and embryo attachment, exhibited large TEPs similar in morphology to those expressed by cultured blastocysts. These observations document for the first time that mammalian blastocysts are capable of endogenous locomotion, confirm TEPs as components of normal blastocyst activity, reveal that there are two kinds of TEPs that differ temporally and morphologically, and extend earlier reports of TEP activity in guinea-pig embryos to the hamster. PMID:8901054

Gonzales, D S; Boatman, D E; Bavister, B D

1996-04-01

192

An oocyte-specific ELAVL2 isoform is a translational repressor ablated from meiotically competent antral oocytes.  

PubMed

At the end of the growth phase, mouse antral follicle oocytes acquire full developmental competence. In the mouse, this event is marked by the transition from the so-called non-surrounded nucleolus (NSN) chromatin configuration into the transcriptionally quiescent surrounded nucleolus (SN) configuration, which is named after a prominent perinucleolar condensed chromatin ring. However, the SN chromatin configuration alone is not sufficient for determining the developmental competence of the SN oocyte. There are additional nuclear and cytoplamic factors involved, while a little is known about the changes occurring in the cytoplasm during the NSN/SN transition. Here, we report functional analysis of maternal ELAVL2 an AU-rich element binding protein. Elavl2 gene encodes an oocyte-specific protein isoform (denoted ELAVL2), which acts as a translational repressor. ELAVL2 is abundant in fully grown NSN oocytes, is ablated during the NSN/SN transition and remains low during the oocyte-to-embryo transition (OET). ELAVL2 overexpression during meiotic maturation causes errors in chromosome segregation, indicating the significance of naturally reduced ELAVL2 levels in SN oocytes. On the other hand, during oocyte growth, prematurely reduced Elavl2 expression results in lower yields of fully grown and meiotically matured oocytes, suggesting that Elavl2 is necessary for proper oocyte maturation. Moreover, Elavl2 knockdown showed stimulating effects on translation in fully grown oocytes. We propose that ELAVL2 has an ambivalent role in oocytes: it functions as a pleiotropic translational repressor in efficient production of fully grown oocytes, while its disposal during the NSN/SN transition contributes to the acquisition of full developmental competence. PMID:24553115

Chalupnikova, Katerina; Solc, Petr; Sulimenko, Vadym; Sedlacek, Radislav; Svoboda, Petr

2014-04-01

193

Penetrating craniofacial arrow injury  

PubMed Central

Arrow injuries are an extinct form of injury in most parts of the developed world, but are still seen, albeit infrequently in developing countries. Reports of penetrating injuries of the craniofacial region secondary to projectiles are few and far between. The morbidity-free outcome of surgical removal, in case of penetrating arrow injuries, despite the delay in presentation and, moreover, in the emergency surgical practice, are the salient points to be remembered whilst managing such cases, for what the mind knows is what the eyes see and what the eyes see is what can be practiced. We report the case of a patient who was attacked by a projectile fired from a crossbow. Immediate surgery under general anesthesia was required to remove the arrow, with utmost care to avoid any neurovascular compromise to the facial nerve, as well as minimize postoperative complications such as otitis media and subsequent meningitis.

Jain, DK; Aggarwal, Gaurav; Lubana, PS; Moses, Sonia

2010-01-01

194

Mars penetrator: Subsurface science mission  

NASA Technical Reports Server (NTRS)

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.

Lumpkin, C. K.

1974-01-01

195

Penetration of yawed projectiles  

Microsoft Academic Search

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°(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

Reaugh

1990-01-01

196

Cyclooxygenase-2 Expression in Hamster and Human Pancreatic Neoplasia1  

PubMed Central

Abstract Cyclooxygenase-2 (COX-2) has been implicated in the development of gastrointestinal malignancies. The aim of the present study was to determine COX-2 expression/activity throughout stages of experimental and human pancreatic neoplasia. COX-2 immunohistochemistry was performed in pancreata of hamsters subjected to the carcinogen N-nitrosobis-(2-oxopropyl)amine (BOP) and in human pancreatic tumors. COX-2 activity was determined by prostaglandin E2 assay in tumor versus matched normal pancreatic tissues. The activity of the COX inhibitor sulindac was tested in the PC-1 hamster pancreatic cancer model. COX-2 expression was elevated in all pancreatic intraepithelial neoplasias (PanINs) and adenocarcinomas. In BOP-treated hamsters, there were significant progressive elevations in COX-2 expression throughout pancreatic tumorigenesis. In human samples, peak COX-2 expression occurred in PanIN2 lesions and remained moderately elevated in PanIN3 and adenocarcinoma tissues. COX-2 activity was significantly elevated in hamster and human pancreatic cancers compared to pair-matched normal pancreas. Furthermore, hamster pancreatic tumor engraftment/formation in the PC-1 hamster pancreatic cancer model was reduced 4.9-fold by oral administration of sulindac. Increased COX-2 expression is an early event in pancreatic carcinogeneses. The BOP-induced hamster carcinogenesis model is a representative model used to study the role of COX-2 in well-differentiated pancreatic tumorigenesis. COX inhibitors may have a role in preventing tumor engraftment/formation.

Yip-Schneider, Michele T; Savage, Jesse J; Hertzler, Dean A; Cummings, William O

2006-01-01

197

Single sperm cryopreservation on cryoloops: an alternative to hamster zona for freezing individual spermatozoa.  

PubMed

Methodology enabling cryopreservation of individual spermatozoa in extreme cases of oligozoospermia would tremendously benefit patients. This study explores the use of a nylon loop for cryopreservation of small quantities of spermatozoa, and also describes a novel technique for freezing individual spermatozoa with this cryoloop. Experiments were conducted to compare sperm recovery and viability after cryopreservation in conventional vials versus on the cryoloops. Discarded human sperm specimens with varying parameters were utilized. The study also examines two different glycerolbased cryoprotectants, with and without test yolk buffer. For single sperm cryopreservation, 5-10 spermatozoa were selected and loaded onto cryoloops with the aid of a microscope and micromanipulation equipment. Sperm function testing was performed on both human and bovine spermatozoa frozen on cryoloops. Microquantities of spermatozoa frozen on cryoloops exhibited overall motility and viability parameters similar to control samples frozen in cryovials. Individually selected spermatozoa cryopreserved on loops were easily recovered and post-thaw motility was generally good. Sperm function testing demonstrated that both human and bovine spermatozoa cryopreserved on loops were able to undergo sperm head decondensation when injected into oocytes. Cryoloops may be an excellent alternative to hamster zonae for cryopreserving small numbers of human spermatozoa. PMID:15257818

Desai, Nina N; Blackmon, Heather; Goldfarb, James

2004-07-01

198

Cryopreservation of embryos and oocytes: an update.  

PubMed

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

Gelety, T; Surrey, E

1993-10-01

199

Oocyte donors experiences of altruistic known donation: a qualitative study  

Microsoft Academic Search

Objective: The purpose of this qualitative study was to investigate the experiences of women who had donated oocytes to a known recipient. Background: Altruistic known donation between friends or family members is the predominant form of oocyte donation in Canada due to legal prohibition of donor compensation. Methods: Data were collected from a hospital-based IVF clinic located in a Canadian

Samantha Yee; Eric Blyth; A. Ka Tat Tsang

2011-01-01

200

Factors affecting the survivability of bovine oocytes vitrified in droplets  

Microsoft Academic Search

Vitrification of bovine oocytes performed using the traditional, in straw system has not given satisfactory results. Although an alternative approach based on minimizing the volume of the vitrified sample has recently resulted in a much more promising survival rate of vitrified oocytes, we attempted to examine some additional factors influencing the survival and subsequent fertilization and development rates of bovine

K. Papis; M. Shimizu; Y. Izaike

2000-01-01

201

The structure of the chromosomes in human primordial oocytes  

Microsoft Academic Search

Primordial oocytes (oocytes in primordial follicles) from human ovaries aged 51\\/2 months post conception to 11 3\\/4 years post partum were examined in: (a) squash preparations of fresh and fixed tissue; (b) histological preparations; and (c) thin sections by electron microscopy, in order to study the structure of the chromosomes. The light microscope shows that the chromosome consists of

T. G. Baker; L. L. Franchi

1967-01-01

202

Total fertilization failure and molecular abnormalities in metaphase II oocytes  

Microsoft Academic Search

Total fertilization failures (TFF) are rare events of IVF by intracytoplasmic sperm injection (ICSI). When male factor is excluded, the lack of identifiable aetiological criteria raises the question of the reliable clinical management. The goal of this study was to identify molecular abnormalities in metaphase II (MII) oocytes yielding TFF. The nuclear mature MII oocytes mRNA expression profiles were compared

Stphan Gasca; Lionel Reyftmann; Franck Pellestor; Thierry Rme; Said Assou; Tal Anahory; Herv Dechaud; Bernard Klein; John De Vos; Samir Hamamah

2008-01-01

203

Human oocyte cryopreservation: a valid alternative to embryo cryopreservation?  

Microsoft Academic Search

Embryo cryopreservation has become an ethical necessity due to the way human in vitro fertilization (IVF) infertility therapy has developed. Limited embryonic implantation has by necessity driven IVF therapy to adopt ways to maximize the harvest of oocytes following ovarian hyperstimulation with its attendant risks. Collection of more oocytes has allowed more embryos to be generated to compensate for poor

Michael Tucker; Paula Morton; Juergen Liebermann

2004-01-01

204

Mural granulosa cell gene expression associated with oocyte developmental competence  

Microsoft Academic Search

BACKGROUND: Ovarian follicle development is a complex process. Paracrine interactions between somatic and germ cells are critical for normal follicular development and oocyte maturation. Studies have suggested that the health and function of the granulosa and cumulus cells may be reflective of the health status of the enclosed oocyte. The objective of the present study is to assess, using an

Jin-Yi Jiang; Huiling Xiong; Mingju Cao; Xuhua Xia; Marc-Andre Sirard; Benjamin K Tsang

2010-01-01

205

Anastral spindle assembly and ?-tubulin in Drosophila oocytes  

Microsoft Academic Search

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

Sharyn A Endow; Mark A Hallen

2011-01-01

206

The 'vanishing embryo' phenomenon in an oocyte donation programme  

Microsoft Academic Search

BACKGROUND: We studied the incidence of vanishing embryos (VE) in pregnancies achieved by oocyte donation and evaluated the obstetric and perinatal complications. METHOD: A retrospective study was carried out based on a chart review of 399 patients with multiple pregnancies from our oocyte donation programme. We defined vanishing phenomenon as the early resorption, in the first trimester, of one or

Manuel Rodr guez-Gonzalez; Vicente Serra; Juan Antonio Garcia-Velasco; Antonio Pellicer

2002-01-01

207

Role of Fyn kinase in oocyte developmental potential  

PubMed Central

Fyn kinase is highly expressed in oocytes, with inhibitor and dominant-negative studies suggesting a role in the signal transduction events during egg activation. The purpose of the present investigation was to test the hypothesis that Fyn is required for calcium signaling, meiosis resumption and pronuclear congression using the Fyn-knockout mouse as a model. Accelerated breeding studies revealed that Fyn-null females produced smaller litter sizes at longer intervals and exhibited a rapid decline in pup production with increasing age. Fyn-null females produced a similar number of oocytes, but the frequency of immature oocytes and mature oocytes with spindle chromosome abnormalities was significantly higher than in controls. Fertilized Fyn-null oocytes frequently (24%) failed to undergo pronuclear congression and remained at the one-cell stage. Stimulation with gonadotropins increased the number of oocytes ovulated, but did not overcome the above defects. Fyn-null oocytes overexpressed Yes kinase in an apparent effort to compensate for the loss of Fyn, yet still exhibited an altered pattern of protein tyrosine phosphorylation. In summary, Fyn-null female mice exhibit reduced fertility that appears to result from actin cytoskeletal defects rather than calcium signaling. These defects cause developmental arrest during oocyte maturation and pronuclear congression.

Luo, Jinping; McGinnis, Lynda K.; Kinsey, William H.

2014-01-01

208

Reprogramming of Human Somatic Cells Using Human and Animal Oocytes  

Microsoft Academic Search

There is renewed interest in using animal oocytes to reprogram human somatic cells. Here we compare the re- programming of human somatic nuclei using oocytes obtained from animal and human sources. Comparative analysis of gene expression in morula-stage embryos was carried out using single-embryo transcriptome am- plification and global gene expression analyses. Genomic DNA fingerprinting and PCR analysis confirmed that

Young Chung; Colin E. Bishop; Nathan R. Treff; Stephen J. Walker; Vladislav M. Sandler; Sandy Becker; Irina Klimanskaya; Wan-Song Wun; Randall Dunn; Rebecca M. Hall; Jing Su; Shi-Jiang Lu; Marc Maserati; Young-Ho Choi; Richard Scott; Anthony Atala; Ralph Dittman; Robert Lanza

2009-01-01

209

Implications of polycystic ovary syndrome on oocyte development.  

PubMed

Human follicle development requires the recruitment of primordial follicles into a cohort of growing follicles from which one follicle is selected to ovulate a mature oocyte. During this developmental process, complex endocrine and intraovarian paracrine signals create a changing intrafollicular hormonal milieu. With this microenvironment, appropriate cumulus cell-oocyte signaling governs oocyte developmental competence, defined as the ability of the oocyte to complete meiosis and undergo fertilization, embryogenesis, and term development. Many of these mechanisms are perturbed in polycystic ovary syndrome (PCOS), a heterogeneous syndrome characterized by ovarian hyperandrogenism, hyperinsulinemia from insulin resistance, and reduced fecundity. In addition to these endocrinopathies, PCOS also is characterized by paracrine dysregulation of follicle development by intraovarian proteins of the transforming growth factor-beta family. Consequently, PCOS patients undergoing ovarian stimulation for in vitro fertilization are at increased risks of impaired oocyte developmental competence, implantation failure, and pregnancy loss. Recent data demonstrate links between endocrine/paracrine factors and oocyte gene expression in PCOS and suggest that new clinical strategies to optimize developmental competence of PCOS oocytes should target correction of the entire follicle growth and oocyte development process. PMID:18181083

Dumesic, Daniel A; Abbott, David H

2008-01-01

210

Mitochondrial DNA deletions in rhesus macaque oocytes and embryos  

Microsoft Academic Search

Mitochondria are the most abundant organelles in mammalian oocytes and early embryos. Mitochondrial DNA (mtDNA) muta- tions, including the common deletion, have been found in skeletal muscle fibres from aged rhesus macaques. The specific aims of this study were to determine whether the mitochondrial common deletion is present in rhesus oocytes after hormonal stimulation and in embryos generated by in

T. C. Gibson; H. M. Kubisch; C. A. Brenner

2005-01-01

211

In vitro growth and maturation of vitrified-warmed bovine oocytes collected from early antral follicles.  

PubMed

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

Hirao, Yuji; Somfai, Tams; Naruse, Kenji

2014-03-01

212

[Thymectomy of adult mice and hamsters].  

PubMed

The author describes a method for thymectomy of adult mice and hamsters, in which he uses a new operative approach at the initial stage of the operation. The common longitudinal median skin section is replaced with semioval section of the skin which begins from the surface transversly to the median line over xiphoid process of the chest bone, passes parallely and lateralily to both sides of the chest bone in the direction to the neck and ends at the level of the clavicle. Semioval cut skin, returned to its place after comrlition of the thymectomy, forms a peculiar subcutaneous pocket in the cavity of which the mediastimum and the risk of pneumothorax, while the usage of skin sutures is replaced completely by the tissue glue. PMID:891449

Kostadinov, A

1977-01-01

213

A role for glucose in hypothermic hamsters  

NASA Technical Reports Server (NTRS)

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.

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

1976-01-01

214

Specific activation requirements of in vitro-matured sheep oocytes following vitrification-warming.  

PubMed

Oocyte vitrification and assisted oocyte activation have increasingly important roles in assisted reproductive technology. Yet, an important area of concern with matured oocyte cryobiology is that elements of oocytes intimately involved in metaphase-II arrest may be modified by cryopreservation. By comparing different cellular characteristics of unvitrified, vitrified-warmed, and unvitrified-activated oocytes, the present study investigated how vitrification-warming process may affect developmental competence of in vitro-matured sheep oocytes following parthenogenetic activation. Structural, ultrastructural, and molecular analyses indicated that the characteristics of vitrified-warmed oocytes vastly differed from fresh oocytes, instead resembling unvitrified-activated oocytes. For unvitrified oocytes, the highest blastocyst yield (41.8??0.6%) was achieved using the maximum ionomycin concentration (5?M), and importantly, the duration of ionomycin treatment was not of utmost importance at this concentration. In contrast, the maximum blastocyst yield of vitrified-warmed oocytes (28.4??1.4%) was achieved with a minimal duration of ionomycin treatment (1?min), and further extending the duration dramatically reduced developmental potential of vitrified-warmed oocytes. These results suggested that vitrified-warmed oocytes may need an activation protocol different from unvitrified oocytes. In this respect, unvitrified oocytes were more sensitive to the concentration rather than the duration of ionomycin treatment when compared with vitrified oocytes, which were sensitive to the treatment duration. These results may provide a platform to improve the potential applications of vitrified oocytes in medicine and agriculture. PMID:22565663

Asgari, V; Hosseini, S M; Ostadhosseini, S; Hajian, M; Azhdari, Z T; Mosaie, M; Nasr-Esfahani, M H

2012-07-01

215

Penetrating Fire Extinguisher  

NASA Technical Reports Server (NTRS)

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.

1985-01-01

216

High survival rate of unfertilized mouse oocytes after vitrification.  

PubMed

Unfertilized mouse oocytes were cooled rapidly by directly plunging them into liquid nitrogen, immediately after exposure to a highly concentrated solution (modified VS1: 2.53 M-dimethyl sulphoxide, 2.36 M-acetamide, 1.19 M-propylene glycol, 5.4% (w/v) polyethylene glycol (Mr 8000) in PB1), and later warmed in a 37 degree C waterbath. After warming, 305 out of 348 oocytes (87.6%) were morphologically normal. After fertilization in vitro of cryopreserved oocytes, the proportions of pronuclear oocytes and 2-cell embryos 5 and 24 h after insemination were 81.6% (124/152) and 78.4% (120/153), respectively. All 2-cell embryos obtained from cryopreserved oocytes were transferred to the oviducts of pseudopregnant recipients and 45.8% (55/120) developed to normal young. PMID:2600904

Nakagata, N

1989-11-01

217

Respiratory Tract Carcinogenesis Induced by Radionuclides in the Syrian Hamster.  

National Technical Information Service (NTIS)

Syrian hamsters were exposed to lung irradiation by various modalities that differed in degree of localization and the fraction of lung exposed. The animals were given alpha emitters under several exposure conditions: intratracheal (IT) instillation of ex...

D. M. Smith R. G. Thomas E. C. Anderson

1979-01-01

218

Karyotype Analysis of Chinese Hamster Chromosomes by Flow Microfluorometry.  

National Technical Information Service (NTIS)

Chromosomes from diploid and aneuploid Chinese hamster cells were isolated at neutral pH or following fixation in 50% acetic acid, stained with ethidium bromide, and analyzed by flow microfluorometry (FMF). In some experiments, the chromosomes were partia...

L. L. Deaven E. Stubblefield J. H. Jett

1976-01-01

219

Bleomycin-stimulated hamster alveolar macrophages release interleukin-1.  

PubMed Central

The capacity of alveolar macrophages (AM) of bleomycin-instilled hamsters to proliferate mouse thymocytes (interleukin-1 activity) and hamster fibroblasts (fibroblast proliferation (FP) activity was studied. Using bleomycin-instilled hamsters, the FP activity of AM culture supernatants was increased significantly on days 1, 5, and 10 after instillation of bleomycin. The interleukin-1 (IL-1) activity, however, was increased significantly on day 1 only as compared with saline-treated hamsters. Next, normal AM were stimulated in vitro by bleomycin. After being fractionated by chromatography, their culture supernatant showed IL-1 activity, which also indicated FP activity. These results suggest that bleomycin directly stimulates AM to release IL-1 in the fibrogenic responses.

Suwabe, A.; Takahashi, K.; Yasui, S.; Arai, S.; Sendo, F.

1988-01-01

220

Nitric oxide and meiotic competence of porcine oocytes.  

PubMed

Reproductive biotechnology such as in vitro fertilization, the creation of transgenic animals or cloning by nuclear transfer depends on the use of fully grown, meiotically competent oocytes capable of completing meiotic maturation by reaching the stage of metaphase II. However, there exists only a limited quantity of these oocytes in the ovaries of females. In view of their limited number, growing oocytes without meiotic competence represent a possible source. The mechanisms controlling the acquisition of meiotic competence, however, are still not completely clear. A gas with a short half-life, nitric oxide (NO), produced by NO-synthase (NOS) enzyme can fulfill a regulatory role in this period. The objective of this study was to ascertain the role of NO in the growth phase of pig oocytes and its influence on the acquisition of meiotic competence with the help of NOS inhibitors, NO donors and their combinations. We demonstrated that the selective competitive iNOS inhibitor aminoguanidine and also the non-selective NOS inhibitor l-NAME block meiotic maturation of oocytes with partial or even full meiotic competence at the very beginning. NOS inhibitors influence even competent oocytes in the first stage of meiotic metaphase. However, blockage is less effective than at the beginning of meiotic maturation. The number of parthenogenetically activated competent oocytes greatly increased in a pure medium after inhibitor reversion. A large quantity of NO externally added to the in vitro cultivation environment disrupts the viability of oocytes. The effectiveness of the inhibitor can be reversed in oocytes by an NO donor in a very low concentration. However, the donor is not capable of pushing the oocytes farther than beyond the first stage of meiotic metaphase. The experiments confirmed the connection of NO with the growth period and the acquisition of meiotic competence. However, it is evident from the experiments that NO is not the only stimulus controlling the growth period. PMID:22440285

Tichovsk, H; Petr, J; Chmelkov, E; Sedmkov, M; T?mov, L; Krej?ov, M; Drflerov, A; Rajmon, R

2011-08-01

221

Effect of carbon tetrachloride on hamster tracheal epithelial cells  

Microsoft Academic Search

This study was designed to assess effects of carbon tetrachloride (CCl4) in hamster tracheal epithelium. Adult, male, Syrian golden hamsters were treated with 2.5 ml\\/kg CCl4 ip, and controls received only the vehicle (peanut oil). Animals were sacrificed after 1, 4, 12, and 24 h. Tissue samples from upper and lower tracheal levels were fixed and embedded in glycol methacrylate

M. Ahmadizadeh; R. Echt; W. W. Heusner; L. M. Ross; R. A. Roth

1990-01-01

222

Isoflavone excretion phenotypes influence plasma cholesterol in golden Syrian hamsters  

Microsoft Academic Search

We hypothesized that hamsters sorted into high and low isoflavone excreter phenotypic categories would show significant differences in plasma cholesterol status, the high isoflavone excreters having lower cholesterol. One-year-old hamsters were fed either 1.18 mmol of total isoflavones per kilogram diet (5 males and 5 females) or 1.77 mmol of total isoflavones per kilogram diet (5 males and 4 females)

Mathieu Renouf; Sun-Ok Lee; Suzanne Hendrich

2006-01-01

223

Circadian Rhythms of Photorefractory Siberian Hamsters Remain Responsive to Melatonin  

Microsoft Academic Search

Short day lengths increase the duration of nocturnal melatonin (Mel) secretion, which induces the winter phenotype in Siberian hamsters. After several months of continued exposure to short days, hamsters spontaneously revert to the spring-summer phenotype. This transition has been attributed to the development of refractoriness of Mel-binding tissues, including the suprachiasmatic nucleus (SCN), to long-duration Mel signals. The SCN of

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

2008-01-01

224

Syrian Hamster-human somatic cell hybrids: Isolation and characterization  

Microsoft Academic Search

Cultured Syrian hamster and human fibroblasts were hybridized by means of Sendai virus. The hybrids were selected chemically. They show preferential loss of human chromosomes and biochemical markers. Therefore they can be used for gene linkage and localization studies in man. Hybrids between Syrian hamster and fibroblasts of a human translocation carrier (46,X,t(14q+,Xq-)) enabled the assignment of the human nucleoside

Karl-Heinz Grzeschik

1973-01-01

225

Evidence for a metabolic limitation of survival in hypothermic hamsters.  

NASA Technical Reports Server (NTRS)

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.

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

1972-01-01

226

Cytogenetic Analysis of Sperm Nucleous Components of Iranian Normal and Sub-Fertile Individuals Using Zona Free Hamster Oocytes  

Microsoft Academic Search

Purpose: The purpose of this study was to investigate whether infertility is affected by sperm chromatin and cytogenetic abnormalities. To this purpose, the frequency of sperm premature chromosome condensation (PCC) induction and numerical chromosome abnormalities in the sperm of normal and sub-fertile men were analyzed. PCC rate was studied for evaluating the role of sperm chromatin abnormalities in the process

H. Mozdarani; A. Mohseni. Meybodi

2004-01-01

227

Fusome asymmetry and oocyte determination in Drosophila.  

PubMed

Germline cysts containing 16 interconnected cells (cystocytes) are produced at an early stage of Drosophila oogenesis by progenitor cells known as cystoblasts that undergo four synchronous rounds of incomplete division. During cyst formation, a region of specialized, spectrin-rich cytoplasm called the fusome traverses the intercellular connections (ring canals), linking individual cystocytes. Subsequently, 15 cystocytes begin to transport specific RNAs and other components into the remaining cell, the future oocyte. We used fusome-specific antibodies to characterize the early stages of cyst formation. During the first cystoblast division, a spherical mass of fusome material (the "spectrosome") was associated with only one pole of the mitotic spindle, revealing that this division is asymmetric. During the subsequent three divisions, the growing fusome always associated with the pole of each mitotic spindle that remained in the mother cell, and only extended through the newly formed ring canals after each division was completed. These observations suggest that fusomes help establish a system of directional transport between cystocytes that underlies oocyte determination. PMID:7758245

Lin, H; Spradling, A C

1995-01-01

228

Effects of bull and heparin and sperm concentrations on in vitro fertilization of buffalo (Bubalus bubalis ) oocytes matured in vitro.  

PubMed

A study was undertaken to assess the ability of spermatozoa from 6 buffalo bulls, at different levels of heparin and sperm concentrations, to achieve an acceptable level of fertilization in vitro. Frozen-thawed spermatozoa, 3 dosages of heparin (0, 10 and 100 ug/ml) in the presence and absence of penicillamine, hypotaurine and epinephrine (PHE), and 4 sperm concentrations (1 x 10(6), 2 x 10(6), 3 x 10(6) and 4 x 10(6) /ml) were studied using 3202 buffalo oocytes. The mean proportions of fertilized oocytes in the group treated with 10 ug/ml of heparin were significantly higher (P<0.05) with the semen of Bulls A, B and C (44.7 to 64.3%) than in medium devoid of heparin. An increase in the dosage of heparin from 10 ug/ml to 100 ug/ml reduced the overall fertilization rate. However, optimal fertilization (30.9%) at 100 ug/ml heparin was observed for semen from Bull D. Bulls E and F yielded the lowest fertilization rate (9.6 and 14.2%, respectively) at the above mentioned heparin dosage. Analysis of sperm density revealed that a concentration of 2 x 10(6) spermatozoa yielded optimal fertilization rates in vitro. Higher sperm concentrations (3 x 10(6) or 4 x 10(6)) resulted in higher oocyte penetration rates but gave rise to polyspermy. PMID:16727261

Totey, S M; Pawshe, C H; Singh, G P

1993-04-01

229

Analysis of factors associated with multiple pregnancy in an oocyte donation programme  

Microsoft Academic Search

The aim of this study is to identify the factors associated with multiple pregnancy in an oocyte donation programme. A retrospective study (20002007) of 945 synchronous cycles was performed. Two embryos were transferred in all cycles on day 2 after oocyte retrieval. All variables (egg donor and recipient age, number of inseminated oocytes, fertilized oocytes, cleaved embryos, good-quality embryos available,

Elisabet Clua; Rosa Tur; Buenaventura Coroleu; Montse Boada; Pere N. Barri; Anna Veiga

2010-01-01

230

Mitochondrial patterns in bovine oocytes with different meiotic competence related to their in vitro maturation.  

PubMed

This study was designed to specify chromatin and mitochondrial patterns in bovine oocytes with different meiotic competence in relation to maturation progress, resumption of meiosis, MII onset and completion of maturation. Oocytes with greater or lesser meiotic competence, recovered separately from medium (MF) and small follicles (SF), were categorized according to morphology. Four oocyte categories, healthy and light-atretic MF and healthy and light-atretic SF oocytes were matured and collected at 0, 3, 7, 16 and 24h of maturation. Specific differences in terms of chromatin and mitochondrial patterns were found among the maturing oocyte categories. Resumption of meiosis was accelerated in light-atretic oocytes, as compared with healthy oocytes, regardless of their meiotic competence. More competent oocytes activated mitochondria twice during maturation, before resumption of meiosis and before completion of maturation, while less competent oocytes did it only once, before completion of maturation. Changes in mitochondrial activity differed in light-atretic compared with healthy in both more and less competent oocytes. Healthy meiotically more competent oocytes formed clusters and produced ATP for the whole time of maturation until its completion, while light-atretic more competent oocytes and healthy less competent oocytes reduced these activities earlier, at MII onset. Contrary to these oocyte categories, light-atretic less competent oocytes increased cluster formation significantly before resumption of meiosis. It can be concluded that bovine oocytes with different meiotic competence and health differed in the kinetics of mitochondrial patterns during maturation. PMID:24716726

Jeseta, M; Ctvrtlikova Knitlova, D; Hanzalova, K; Hulinska, P; Hanulakova, S; Milakovic, I; Nemcova, L; Kanka, J; Machatkova, M

2014-06-01

231

Advanced ground penetrating radar  

SciTech Connect

An advanced Ground Penetrating Radar (GPR) system has the potential for efficiently and reliably providing high resolution images for inspecting concrete civil structures for defects and damage assessment. To achieve the required performance, improvements in radar hardware, and development and adaptation of advanced 2- and 3-dimensional synthetic aperture imaging techniques are needed. Recent and continuing advancement in computer and computer-related technology areas have made it possible to consider more complex and capable systems for a variety of imaging applications not previously conceived. The authors developed conceptual designs, analyzed system requirements, and performed experiments, modeling, and image reconstructions to study the feasibility of improving GPR technology for non-destructive evaluation of bridge decks and other high-value concrete structures. An overview and summary of practical system concepts and requirements, are presented.

Warhus, J.P.; Mast, J.E.; Johansson, E.M.; Nelson, S.D. [Lawrence Livermore National Lab., CA (United States). Electronics Engineering Dept.

1994-07-26

232

Histone deacetylase induces accelerated maturation in Xenopus laevis oocytes.  

PubMed

In oocyte maturation in Xenopus laevis, nuclear material induces rapid maturation and is required for entry into meiosis II. Nuclear material contains a large number of RNAs and proteins, including histone deacetylase (HDAC); however, it is not known which materials induce accelerated maturation. The HDAC activity modifies transcription rate and is required for normal meiosis; however, its function in oocyte maturation is still unclear. We investigated the function of HDAC activity, which is localized in the nuclear material, in the regulation of the speed of oocyte maturation. Inhibition of HDAC activity with trichostatin A (TSA) induced hyperacetylation of histone H3 and prolonged oocyte maturation. In contrast, increase in HDAC activity with an injection of FLAG-tagged maternal histone deacetylase (HDACm-FLAG) mRNA induced deacetylation of histone H3 and reduced the duration of oocyte maturation. Cdc2 kinase, Cdc25C or mitogen-activated protein kinase (MAPK), which are key regulators of the meiosis, were activated coincidently with maturation progression. In oocytes, the mRNA level of Cdc25C, an activator of Cdc2, was increased by HDACm-FLAG mRNA-injection; in contrast, the mRNA level of Cdc2 inhibitor Wee1 was increased by TSA treatment. These results suggest that HDAC activity is involved in the control of maturation speed through the regulation of mRNA levels of cell cycle regulators. Thus, HDACm is a candidate for the nuclear material component that induces rapid maturation in Xenopus oocytes. PMID:23346879

Iwashita, Jun; Kodama, Ayumi; Konno, Yuuri; Abe, Tatsuya; Murata, Jun

2013-04-01

233

Analysis of oocyte physiology to improve cryopreservation procedures.  

PubMed

In contrast to the preimplantation mammalian embryo, it has been notoriously difficult to cryopreserve the metaphase II oocyte. The ability to store oocytes successfully at -196 degrees C has numerous practical and financial advantages, together with ethical considerations, and will positively impact animal breeding programs and assisted conception in the human. Differences in membrane permeability and in physiology are two main reasons why successful oocyte cryopreservation has remained elusive. It is proposed, therefore, that rather than relying on technologies already established for the preimplantation embryo, the development of cryopreservation techniques suitable for the mammalian oocyte needs to take into account the idiosyncratic physiology of this cell. Analysis of intracellular calcium, for example, has revealed that exposure to conventional permeating cryoprotectants, such as propanediol, ethylene glycol and DMSO, all independently result in an increase in calcium, which in turn has the potential to initiate oocyte activation, culminating in zona hardening. Quantification of the metabolome and proteome of the oocyte has revealed that whereas slow freezing has a dramatic effect on cell physiology, vitrification appears to have limited effect. This is plausibly achieved by the limited exposure to cryoprotectants. Analysis of meiotic spindle dynamics and embryo development following IVF, also indicate that vitrification is less traumatic than slow freezing, and therefore has the greatest potential for successful oocyte cryopreservation. PMID:17049589

Gardner, David K; Sheehan, Courtney B; Rienzi, Laura; Katz-Jaffe, Mandy; Larman, Mark G

2007-01-01

234

Oocyte Maturation: The Coming of Age of a Germ Cell  

PubMed Central

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

Jamnongjit, Michelle; Hammes, Stephen R.

2006-01-01

235

Lipofuscin bodies in human oocytes as an indicator of oocyte quality.  

PubMed

Refractile bodies are one of the main morphological abnormalities that can be observed in the cytoplasm of human oocytes. In the present studies the characteristics of refractile bodies and the relationship between the size of these structures and developmental competence of the affected oocytes and resulting embryos were examined. The refractile bodies were found to have yellow autofluorescence which was consistent with the typical autofluorescence of lipofuscin. Viewed by transmitted electron microscopy, the refractile bodies showed the conventional morphology of lipofuscin inclusions and consisted of a mixture of lipids and dense granule materials. Large refractile bodies (>5 microm) were positively stained by the Schmorl reaction and were considered to contain lipofuscin. These larger lipofuscin inclusions (>5 microm) were associated with significantly reduced fertilization and unfavorable blastocyst development. PMID:17653849

Otsuki, Junko; Nagai, Yasushi; Chiba, Kazuyoshi

2007-07-01

236

Embryonic development after intra-follicular transfer of horse oocytes.  

PubMed

A technique was developed in which immature horse oocytes, obtained from slaughterhouse specimens, were transferred to the pre-ovulatory follicle of a mare in vivo, with resulting oocyte maturation, ovulation, fertilization and embryo development. Oocytes were collected from all follicles greater than 3 mm, and were classified as immature, maturing, expanded or denuded. The transfers were performed in the standing, tranquilized mare. The ovary containing the pre-ovulatory follicle was grasped per rectum. A trochar and cannula were placed through the abdominal wall in the flank area, ipsilateral to the grasped ovary. For the transfer, the operator introduced a needle through the cannula to puncture the outer wall of the follicle, while adjusting the position of the ovary per rectum. The mares were inseminated the day after transfer. Twenty oocyte transfers were performed. In 1 mare, 15 immature oocytes were transferred to the pre-ovulatory follicle and 12 oocytes with expanded cumuli were recovered from the follicle 24 h later. In 3 of the remaining 19 mares, the follicle filled with blood after the transfer and ovulation did not occur. Sixteen mares ovulated after oocyte transfer. One mare was killed 3 days following ovulation; flush of the removed oviduct yielded 1 embryo, 2 recently ovulated oocytes and 3 degenerating oocytes. A uterine flush for embryo recovery was performed in each of the other 15 ovulating mares, 7-11 days after ovulation. Embryos were recovered in 7 of the 15 flushes, and embryos in excess of the number of ovulations were recovered from 4 mares (2, 3, 4 and 7 embryos). PMID:1795280

Hinrichs, K; DiGiorgio, L M

1991-01-01

237

Involvement of the prostate and testis expression (PATE)-like proteins in sperm-oocyte interaction  

PubMed Central

BACKGROUND The prostate and testis expression (PATE)-like family of proteins are expressed mainly in the male genital tract. They are localized in the sperm head and are homologous to SP-10, the acrosomal vesicle protein also named ACRV1. Our aim was to characterize the expression and functional role of three PATE-like proteins in the testis and ejaculated sperm. METHODS The expression and localization of PATE-like proteins in human testis biopsies (n= 95) and sperm cells were assessed by RTPCR, immunohistochemistry and immunofluorescence staining (at least 600 sperm cells per specimen). The function of the PATE protein was tested by the hemizona assay and hamster egg penetration test (HEPT). RESULTS PATE and PATE-M genes and proteins were present almost exclusively in germ cells in the testis: immunoflourescence showed that the percentage of germ cells positive for PATE, PATE-M and PATE-B was 85, 50 and 2%, respectively. PATE and PATE-M proteins were localized in the equatorial segment of the sperm head, while PATE-B protein was localized in the post-acrosomal region. A polyclonal antibody (Ab, at 1:50 and 1:200 dilutions) against the PATE protein did not inhibit spermzona binding in the hemizona assay (hemizona index of 89.6 10 and 87 36%, respectively). However, there was inhibition of spermoolemma fusion and penetration in the HEPT (penetration index: without Ab 7 3.9; Ab dilution of 1:100, 4 3.5; Ab dilution of 1:20, 0.6 1.2, P < 0.001). CONCLUSIONS Our data suggest that PATE protein is involved in spermoolemma fusion and penetration but not spermzona binding.

Margalit, M.; Yogev, L.; Yavetz, H.; Lehavi, O.; Hauser, R.; Botchan, A.; Barda, S.; Levitin, F.; Weiss, M.; Pastan, I.; Wreschner, D.H.; Paz, G.; Kleiman, S.E.

2012-01-01

238

Mouse embryos generated from frozen-thawed oocytes can successfully survive a second cryopreservation  

Microsoft Academic Search

BACKGROUND: To determine whether mouse embryos generated from frozen-thawed oocytes can successfully survive a second cryopreservation. METHODS: Immature C57BL6*BALB\\/c female mice underwent superovulation and the collected oocytes were divided into three groups. Group A oocytes (n = 107) underwent IVF. Group B oocytes (n = 167) underwent IVF and embryos generated were then cryopreserved. Group C oocytes (n = 94)

Ariel Revel; Naama Moshe; Aharon Helman; Anat Safran; Alex Simon; Moriah Koler

239

MG-132, an inhibitor of proteasomes and calpains, induced inhibition of oocyte maturation and aneuploidy in mouse oocytes  

PubMed Central

Background Although chromosome missegregation during oocyte maturation (OM) is a significant contributor to human morbidity and mortality, very little is known about the causes and mechanisms of aneuploidy. Several investigators have proposed that temporal perturbations during OM predispose oocytes to aberrant chromosome segregation. One approach for testing this proposal is to temporarily inhibit the activity of protein proteolysis during OM. We used the reversible proteasome inhibitor MG-132 to transiently perturb the temporal sequence of events during OM and subsequently analyzed mouse metaphase II (MII) for cytogenetic abnormalities. The transient inhibition of proteasome activity by MG-132 resulted in elevated levels of oocytes containing extra chromatids and chromosomes. Results The transient inhibition of proteasome-mediated proteolysis during OM by MG-132 resulted in dose-response delays during OM and elevated levels of aneuploid MII oocytes. Oocytes exposed in vitro to MG-132 exhibited greater delays during metaphase I (MI) as demonstrated by significantly (p < 0.01) higher levels of MI arrested oocytes and lower frequencies of premature sister chromatid separation in MII oocytes. Furthermore, the proportions of MII oocytes containing single chromatids and extra chromosomes significantly (p < 0.01) increased with MG-132 dosage. Conclusions These data suggest that the MG-132-induced transient delay of proteasomal activity during mouse OM in vitro predisposed oocytes to abnormal chromosome segregation. Although these findings support a relationship between disturbed proteasomal activity and chromosome segregation, considerable additional data are needed to further investigate the roles of proteasome-mediated proteolysis and other potential molecular mechanisms on chromosome segregation during OM.

Mailhes, John B; Hilliard, Colette; Lowery, Mary; London, Steve N

2002-01-01

240

The dormant and the fully competent oocyte: comparing the transcriptome of human oocytes from primordial follicles and in metaphase II.  

PubMed

Oocytes become enclosed in primordial follicles during fetal life and remain dormant there until activation followed by growth and meiotic resumption. Current knowledge about the molecular pathways involved in oogenesis is incomplete. This study identifies the specific transcriptome of the human oocyte in the quiescent state and at the pinnacle of maturity at ovulation. In silico bioinformatic comparisons were made between the transcriptome of human oocytes from dormant primordial follicles and that of human metaphase II (MII) oocytes and granulosa cells and unique gene expression profiles were identified as well as functional and pathway enrichments associated with the oocytes from the two developmental hallmarks. A total of 729 genes were highly enriched in oocytes from primodial follicles and 1456 genes were highly enriched in MII oocytes (>10-fold, P < 0.001) representing functional categories such as cell cycle regulation, DNA protection and epigenetics, with representative genes validated by qPCR analysis. Dominating canonical pathways in the oocytes from primordial follicles were androgen, estrogen receptor, glucocorticoid receptor and PI3K/AKT signaling (P < 0.001). In the MII, mitotic roles of polo-like kinases, estrogen receptor, JAK/Stat signaling (P < 0.001) and the ERK/MAPK (P < 0.01) signaling were enriched. Some of the highly differentially expressed genes were completely new in human reproduction (CDR1, TLC1A, UHRF2) while other genes [ABO, FOLR1 (folate receptor), CHRNA3 (nicotine receptor)] may relate to clinical observations as diverse as premature ovarian failure, folic acid deficiency and smoking affecting female fertility. The in silico analysis identified novel reproduction-associated genes and highlighted molecular mechanisms and pathways associated with the unique functions of the human oocyte in its two extremes during folliculogenesis. The data provides a fundamental basis for future functional studies in regulation of human oogenesis. PMID:23598597

Grndahl, Marie Louise; Borup, Rehannah; Vikes, Jonas; Ernst, Erik; Andersen, Claus Yding; Lykke-Hartmann, Karin

2013-09-01

241

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

PubMed Central

Understanding of the aetiology of congenitally anomalous pulmonary valves remains incomplete. The aim of our study, therefore, was to elucidate the degree to which the phenotypic variation known to exist for the pulmonary valve relies on genotypic variation. Initially, we tested the hypothesis that genetically alike individuals would display similar valvar phenotypes if the phenotypic arrangement depended entirely, or almost entirely, on the genotype. Thus, we examined pulmonary valves from 982 Syrian hamsters belonging to two families subject to systematic inbreeding by crossing siblings. Their coefficient of inbreeding was 0.999 or higher, so they could be considered genetically alike. External environmental factors were standardized as much as possible. A further 97 Syrian hamsters from an outbred colony were used for comparative purposes. In both the inbred and outbred hamsters, we found valves with a purely trifoliate, or tricuspid, design, trifoliate valves with a more or less extensive fusion of the right and left leaflets, bifoliate, or bicuspid, valves with fused right and left leaflets, with or without a raphe located in the conjoined arterial sinus, and quadrifoliate, or quadricuspid, valves. The incidence of the different valvar morphological variants was similar in the outbred and inbred colonies, except for the bifoliate pulmonary valves, which were significantly more frequent in the hamsters from one of the two inbred families. Results of crosses between genetically alike hamsters revealed no significant association between the pulmonary valvar phenotypes as seen in the parents and their offspring. The incidence of bifoliate pulmonary valves, nonetheless, was higher than statistically expected in the offspring of crosses where at least one of the parents possessed a pulmonary valve with two leaflets. Our observations are consistent with the notion that the basic design of the pulmonary valve, in terms of whether it possesses three or two leaflets, relies on genotypic determinants. They also denote that the bifoliate condition of the valve is the consequence of complex inheritance, with reduced penetrance and variable expressivity. Moreover, in showing that the incidence of the bifoliate pulmonary valve significantly differs in two different isogenetic backgrounds, our data suggest that genetic modifiers might be implicated in directing the manifestation of such specific pulmonary valvar malformations. Finally, our findings indicate that factors other than the genotype, operating during embryonic life and creating developmental noise, or random variation, play a crucial role in the overall phenotypic variation involving the pulmonary valve.

Carmen Fernandez, M; Duran, Ana C; Fernandez, Borja; Arque, Josep M; Anderson, Robert H; Sans-Coma, Valentin

2012-01-01

242

Proteomic Analysis of Chinese Hamster Ovary Cells  

PubMed Central

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.

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

243

Percutaneous penetration of hair dyes  

Microsoft Academic Search

Scalp penetration of 7 hair dyes (oxidative and direct) that occurs under conditions of hair dye usage was evaluated for both rhesus monkey and man using 14C labeled materials by quantifying their absorbtion via urine assays. Both species showed a remarkably similar pattern of dye penetration. The extent of scalp penetratoon is slightly higher for direct dyes but in neither

L. J. Wolfram; H. I. Maibach

1985-01-01

244

Normal Fuze Impact and Penetration.  

National Technical Information Service (NTIS)

A model for the penetration through a thin target by a fuzed projectile at normal incidence was developed. It was assumed that the penetration mechanisms were plugging followed by petalling. From this model the force and stress distributions acting on the...

P. Gordon F. Lee M. Schwartz

1975-01-01

245

Mechanics Approach to Projectile Penetration.  

National Technical Information Service (NTIS)

In the program reported in the paper, a mathematical model was developed which describes the mechanism of the normal penetration of metallic targets. The model considers all the forces acting on the projectile during penetration, bearing in mind that it i...

J. Awerbuch

1970-01-01

246

Projectile penetration into representative targets  

Microsoft Academic Search

The differential equation representing the penetration of a 'hard' projectile into semi-infinite, homogeneous target materials is solved for several generic combinations of the target material\\/projectile characteristics. A 'hard' projectile is defined as one that does not change size or shape and does not lose mass during the penetration process. The target materials evaluated range from the structurally 'soft' materials (liquids)

George W. Stone

1994-01-01

247

Sidewall penetrator for oil wells  

NASA Technical Reports Server (NTRS)

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.

Collins, E. R., Jr.

1981-01-01

248

Penetration below a convective zone  

Microsoft Academic Search

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

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

1994-01-01

249

Static penetration resistance of soils  

NASA Technical Reports Server (NTRS)

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.

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

1973-01-01

250

Hypotonicity activates a native chloride current in Xenopus oocytes.  

PubMed

Xenopus oocytes are frequently utilized for in vivo expression of cellular proteins, especially ion channel proteins. A thorough understanding of the endogenous conductances and their regulation is paramount for proper characterization of expressed channel proteins. Here we detail a novel chloride current (ICl.swell) responsive to hypotonicity in Xenopus oocytes using the two-electrode voltage clamp technique. Reducing the extracellular osmolarity by 50% elicited a calcium-independent chloride current having an anion conductivity sequence identical with swelling-induced chloride currents observed in epithelial cells. The hypotonicity-activated current was blocked by chloride channel blockers, trivalent lanthanides, and nucleotides. G-protein, cAMP-PKA, and arachidonic acid signaling cascades were not involved in ICl.swell activation. ICl.swell is distinct from both stretch-activated nonselective cation channels and the calcium-activated chloride current in oocytes and may play a critical role in volume regulation in Xenopus oocytes. PMID:8189203

Ackerman, M J; Wickman, K D; Clapham, D E

1994-02-01

251

Functional expression of murine multidrug resistance in Xenopus laevis oocytes  

SciTech Connect

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.

Castillo, G.; Vera, J.C.; Rosen, O.M. (Memorial Sloan-Kettering Cancer Research Center, New York, NY (USA)); Yang, Chiaping Huang; Horwitz, S.B. (Albert Einstein College of Medicine, Bronx, NY (USA))

1990-06-01

252

Recent advances in oocyte and ovarian tissue cryopreservation and transplantation  

PubMed Central

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.

Rodriguez-Wallberg, Kenny A.; Oktay, Kutluk

2012-01-01

253

Role of Oocyte Loss in Ovarian Surface Mesothelial Cell Transformation.  

National Technical Information Service (NTIS)

Three Specific Aims (SA) were proposed to test in mice if accelerated oocyte loss caused by Bclw deficiency or Bax gain-of-function drives ovarian surface mesothelial cell (OSMC) transformation: (1) characterize preneoplastic changes in OSMC of Bclw(-/-)1...

J. L. Tilly

2003-01-01

254

Role of Oocyte Loss in Ovarian Surface Mesothelial Cell Transformation.  

National Technical Information Service (NTIS)

Three Specific Aims (SA) were proposed to test in mice the hypothesis that accelerated oocyte loss caused by Bclw deficiency or Bax gain-of-function drives ovarian surface mesothelial cell (OSMC) transformation: (1) characterize preneoplastic changes in O...

J. L. Tilly G. R. MacGregor

2002-01-01

255

Saprophytic and cycloheximide resistant fungi isolated from golden hamster.  

PubMed

Healthy hair samples from golden hamsters were examined for the presence of dermatophytes and non-dermatophytes using baiting technique and direct inoculation. Thirty-four species and 2 varieties attributed to 17 genera were recovered. Paecilomyces variotii (isolated from 84.4% of the examined hair) and Aspergillus niger (81.3%) were the more frequent isolates on Sabouraud's dextrose agar (SDA) without cycloheximide. Our results have clearly demonstrated that the hair of hamster was free from true dermatophytes. Using the dilution plate method many fungal species were isolated from cage material (7 genera and 10 species + 1 variety); from faeces (10 genera and 17 species); from standard chow (3 genera and 6 species) of hamster. P. variotii which was the most frequent fungus in the preceding 3 substrates was completely absent in the presence of cycloheximide in SDA. The present study has demonstrated for the first time the isolation of Trichophyton rubrum from hamster faeces. Also, several saprophytic and cycloheximide resistant fungi were isolated. In the air of hamster cage Cladosporium cladosporioides, Penicillium chrysogenum, Alternaria alternata and Scopulariopsis brevicaulis were the most dominant species on SDA with or without cycloheximide. Using the agar diffusion method, Aloe sap, onion oil, garlic bulb extract and aqueous leaf extracts of Andropogon citratus, Euphorbia sp. and Ruta graveolens were tested for their antifungal activity on 10 fungal species. It was observed that onion oil exhibited a high inhibitory effect against most of the tested fungi. PMID:9768288

Bagy, M M; el-Shanawany, A A; Abdel-Mallek, A Y

1998-01-01

256

Circadian regulation of cortisol release in behaviorally split golden hamsters.  

PubMed

The master circadian clock located within the hypothalamic suprachiasmatic nucleus (SCN) is necessary for the circadian rhythm of glucocorticoid (GC) release. The pathways by which the SCN sustains rhythmic GC release remain unclear. We studied the circadian regulation of cortisol release in the behaviorally split golden hamster, in which the single bout of circadian locomotor activity splits into two bouts approximately 12 h apart after exposing the animals to constant light conditions. We show that unsplit control hamsters present a single peak of cortisol release that is concomitant with a single peak of ACTH release. In contrast, split hamsters show two peaks of cortisol release that are approximately 12 h appart and are appropriately phased to each locomotor activity bout but surprisingly do not rely on rhythmic release of ACTH. Our results are consistent with a model in which the circadian pacemaker within the SCN regulates the circadian release of GC via input to the hypothalamo-pituitary-adrenal axis and via a second regulatory pathway, which likely involves sympathetic innervation of the adrenal and can operate even in the absence of ACTH circadian rhythmic release. Furthermore, we show that although the overall 24-h cortisol output in split hamsters is lower than in unsplit controls, split hamsters release constant low levels of ACTH. This result suggests that the timing, rather than the absolute amount, of cortisol release is more critical for the induction of negative feedback effects that regulate the hypothalamo-pituitary-adrenal axis. PMID:22128030

Lilley, Travis R; Wotus, Cheryl; Taylor, Daniel; Lee, Jennifer M; de la Iglesia, Horacio O

2012-02-01

257

Hamster and murine models of severe destructive Lyme arthritis.  

PubMed

Arthritis is a frequent complication of infection in humans with Borrelia burgdorferi. Weeks to months following the onset of Lyme borreliosis, a histopathological reaction characteristic of synovitis including bone, joint, muscle, or tendon pain may occur. A subpopulation of patients may progress to a chronic, debilitating arthritis months to years after infection which has been classified as severe destructive Lyme arthritis. This arthritis involves focal bone erosion and destruction of articular cartilage. Hamsters and mice are animal models that have been utilized to study articular manifestations of Lyme borreliosis. Infection of immunocompetent LSH hamsters or C3H mice results in a transient synovitis. However, severe destructive Lyme arthritis can be induced by infecting irradiated hamsters or mice and immunocompetent Borrelia-vaccinated hamsters, mice, and interferon-gamma- (IFN-?-) deficient mice with viable B. burgdorferi. The hamster model of severe destructive Lyme arthritis facilitates easy assessment of Lyme borreliosis vaccine preparations for deleterious effects while murine models of severe destructive Lyme arthritis allow for investigation of mechanisms of immunopathology. PMID:22461836

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

2012-01-01

258

The Syrian hamster model of hantavirus pulmonary syndrome  

PubMed Central

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

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

2012-01-01

259

Lymphoma outbreak in a GASH:Sal hamster colony.  

PubMed

We have detected a high incidence of lymphomas in a colony of GASH:Sal Syrian golden hamsters (Mesocricetus auratus). This strain is characterised by its ability to present convulsive crises of audiogenic origin. Almost 16 % (90 males and 60 females) of the 975 animals were affected during a 5-year period by the development of a progressing lymphoid tumour and exhibited similar clinical profiles characterised by lethargy, anorexia, evident abdominal distension, and a rapid disease progression resulting in mortality within 1 to 2 weeks. A TaqMan probe-based real-time PCR analysis of genomic DNA from different tissue samples of the affected animals revealed the presence of a DNA sequence encoding the hamster polyomavirus (HaPyV) VP1 capsid protein. Additionally, immunohistochemical analysis using HaPyV-VP1-specific monoclonal antibodies confirmed the presence of viral proteins in all hamster tumour tissues analysed within the colony. An indirect ELISA and western blot analysis confirmed the presence of antibodies against the VP1 capsid protein in sera, not only from affected and non-affected GASH:Sal hamsters but also from control hamsters from the same breeding area. The HaPyV genome that accumulated in tumour tissues typically contained deletions affecting the noncoding regulatory region and adjacent sequences coding for the N-terminal part of the capsid protein VP2. PMID:23719671

Muoz, Luis J; Ludea, Dolores; Gedvilaite, Alma; Zvirbliene, Aurelija; Jandrig, Burkhard; Voronkova, Tatyana; Ulrich, Rainer G; Lpez, Dolores E

2013-11-01

260

Electromagnetic Field Penetration Studies  

NASA Technical Reports Server (NTRS)

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.

Deshpande, M.D.

2000-01-01

261

Structure and characterization of hamster IL12 p35 and p40  

Microsoft Academic Search

Complementary DNAs coding for two subunits of hamster interleukin-12 (IL-12), p35 and p40, were cloned from a hamster dendritic cell (DC) cDNA library. The cloning demonstrated that hamster IL-12 consisted of a p35 subunit with 216 amino acid (aa) residues and a p40 subunit with 327 aa. Structural comparison of hamster p35 and p40 at the protein level showed the

Kouji Maruyama; Yutaka Takigawa; Yasuto Akiyama; Takashi Hojo; Noriko Nara-Ashizawa; Jin-yan Cheng; Morihiro Watanabe; Ken Yamaguchi

2003-01-01

262

PROTEIN SYNTHESIS AND UPTAKE BY ISOLATED CECROPIA OOCYTES  

Microsoft Academic Search

SUMMARY A procedure has been developed for separating the oocytes and follicular epithelium-nurse cell complexes making up the vitellogenic ovarian follicle of the Cecropia moth. Both com- ponents remained viable during short-term in vitro incubation in female blood. Isolated epithelial cells were found by autoradiography to incorporate tritiated amino acids and to secrete a fixable, non-dialysable labelled material. Isolated oocytes

LUCY M. ANDERSON

263

Transvaginal collection and ultrastructure of Llama ( Lama glama ) oocytes  

Microsoft Academic Search

Ultrasound-guided transvaginal follicle aspiration has been described as a noninvasive and repeatable procedure for oocyte collection in several species, but its use has not been described for any of the members of the family, Camelidae. A study was designed to determine the feasibility of an ultrasound-guided transvaginal approach for oocyte collection in llamas. Fifteen non-pregnant, adult female llamas (10 non-stimulated

G. M. Brogliatti; A. T. Palasz; H. Rodriguez-Martinez; R. J. Mapletoft; G. P. Adams

2000-01-01

264

Resveratrol, an effective regulator of ovarian development and oocyte apoptosis.  

PubMed

Resveratrol, a phytopolyphenol compound found chiefly in grapes and wine, has been reported to have a variety of anti-inflammatory, anti-platelet, and anti-carcinogenic effects. However, little is known about the effects of resveratrol on ovarian development and oocyte apoptosis. We investigated the effects of resveratrol on ovarian development in rats with different ages [from post-natal day (PD) 1 to 15 months], as well as on oocyte apoptosis in PD1 and PD2 rat ovaries. We show that: a) ip injection of resveratrol (20 mg/kg/day) increased the percentage of unassembled follicles and the total number of oocytes in PD1 and PD2 rat ovaries. Similar results were obtained when mothers were treated with resveratrol (20 mg/kg/day) by intragastric administration from day 11, after the detection of vaginal plug, until delivery. In PD4 rat ovaries, the total number of oocytes was significantly increased in the groups treated with resveratrol. Moreover, more unassembled follicles and fewer primary follicles were present in the groups treated with resveratrol than in the controls; b) in 15-month-old rat ovaries, resveratrol increased the number of resting follicles and total oocytes, and decreased the number of developing follicles and atretic follicles; 3) the percentage of TUNEL-positive oocytes decreased in PD1 and PD2 rat ovaries after resveratrol treatment, and the number of oocytes positive for Foxo3a, Bim, and p27KIP1 in PD2 rat ovaries was lower in the resveratrol treatment group than in controls. These results suggest that resveratrol may delay oocyte nest breakdown and inhibit both the primordial-to-developing-follicle transition and apoptosis by decreasing the activation of Foxo3a, Bim, and p27KIP1, thus augmenting the resting follicle reserves, maintaining regular estrous cycles of early aged rats and delaying climacterium. PMID:21738004

Kong, X-X; Fu, Y-C; Xu, J-J; Zhuang, X-L; Chen, Z-G; Luo, L-L

2011-12-01

265

OOCYTE DIFFERENTIATION AND VITELLOGENESIS IN THE ROACH PERIPLANETA AMERICANA  

PubMed Central

The ovary of the roach Periplaneta americana has been studied by techniques of light and electron microscopy. Each ovariole (panoistic type) contains a linear array of oocytes in varying stages of development. Newly formed oocytes become encased by a layer of follicle cells and begin pinocytosis. All subsequent growth stages of the oocytes are dependent, in part, on this phenomenon. All of the pinocytotic caveolae show an unique surface modification; i.e., on their internal surface they have an amorphous or filamentous substance and their external surface is studded with many fine radially oriented spike-like projections. The pinosomes of early oocytes do not contain a demonstrable internal structure; they are thought to contain nutritive substances for the developing oocytes rather than yolk precursors. When the oocyte enters its last stage of growth, characterized by yolk deposition, the caveolae become filled with a dense material which is thought to be the precursors of yolk. Hence the conclusion is drawn that yolk formation is independent of any cytoplasmic organelle system of the oocyte and that the precursors of this deutoplasmic substance are manufactured outside the ovary and are internalized by the process of pinocytosis. Under the phase-contrast microscope the nucleoli of early oocytes are large irregular masses and show the phenomenon of nucleolar emission (fragmentation). These "emissions" become randomly dispersed in the nucleoplasm and some of them come to be intimately associated with the fenestrated nuclear envelope. After this process ceases, the main nucleolar mass becomes vacuolated. Electron micrographs suggest that the constituent particles of the nucleolar emissions migrate from the nucleus through patent pores of the nuclear envelope.

Anderson, Everett

1964-01-01

266

C-type natriuretic peptide inhibits porcine oocyte meiotic resumption.  

PubMed

Summary 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

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

2014-08-01

267

Epinephrine promotes development potential of vitrified mouse oocytes.  

PubMed

Cryopreserved oocytes show low developmental ability. To understand the mechanism underlying their development impairment, study was designed to determine the effect of epinephrine on the in vitro developmental competence of vitrified mouse oocytes. Mature oocytes were vitrified using Open Pulled Straw (OPS) method. The vitrified oocytes were warmed and introduced into M2 medium which contains epinephrine at different concentrations (10(-2), 10(-4), 10(-6), 10(-8) mol L(-1) in an incubator for 1 h. Then the survival rate of the oocytes was evaluated and the subsequent development of oocytes was assessed through in vitro Fertilization (IVF). Furthermore, the levels of intracellular ROS, GSH and the concentration of ATP were determined among 10(-4) mol L(-1) epinephrine-treated group, vitrification group and fresh group. Results showed that vitrified oocytes treated with 10(-4)) mol L(-1) epinephrine had significant higher rates of cleavage (66.4 vs.45.2%) and blastocyst (47.2 vs. 34.7%) than no epinephrine treated group, as well as more blastocyst cells (54.5 vs. 36.8) and lower ratio of apoptotic cells (5.9 vs. 21.5%; p < 0.05). Further experiment found that 10(-4) mol L(-1) epinephrine treatment could significantly reduce intracellular ROS level and enhance cytoplasmic ATP concentration (p < 0.05), but there was no different in GSH level compared to vitrification group. In conclusion, epinephrine could promote vitrified oocytes cryosurvival and their subsequent development ability, which maybe related with the changes of intracellular ROS level and ATP content. PMID:24783810

Wang, Liang; Fu, Xiangwei; Zeng, Yan; Zhu, Shien

2014-01-15

268

Highly efficient vitrification method for cryopreservation of human oocytes  

Microsoft Academic Search

Two experiments were performed to develop a method to cryopreserve MII human oocytes. In the first experiment, three vitrification methods were compared using bovine MII oocytes with regard to their developmental competence after cryopreservation: (i) vitrification within 0.25-ml plastic straws followed by in-straw dilution after warming (ISD method); (ii) vitrification in open-pulled straws (OPS method); and (iii) vitrification in <0.1

Masashige Kuwayama; Gbor Vajta; Osamu Kato

2005-01-01

269

Parthenogenetic activation of oocytes in c-mos-deficient mice  

Microsoft Academic Search

IN Xenopus the c-mos proto-oncogene product (Mos) is essential for the initiation of oocyte maturation1, for the progression from meiosis I to meiosis II2,3 and for the second meiotic metaphase arrest, acting as an essential component of the cytostatic factor CSF4,5. Its function in mouse oocytes is unclear6-9, however, as is the biological significance of c-mos mRNA expression in testes1,10

Naohiro Hashimoto; Nobumoto Watanabe; Yasuhide Furuta; Hiroyuki Tamemoto; Noriyuiki Sagata; Minesuke Yokoyama; Kenji Okazaki; Mariko Nagayoshi; Naoki Takedat; Yoji Ikawatll; Shinichi Aizawai

1994-01-01

270

Effect of exogenous gonadotropins on endometrial maturation in oocyte donors  

Microsoft Academic Search

Objective: To determine the effects of controlled ovarian hyperstimulation (COH) on endometrial maturation.Design: Prospective, before and after evaluation of midluteal endometrial biopsies in oocyte donors spontaneous and subsequent COH cycles.Setting: Tertiary academic medical center assisted reproductive technologies clinic.Patient(s): Nineteen oocyte donors.Intervention(s): Exogenous gonadotropins, endometrial biopsies.Main Outcome Measure(s): Endometrial histology and an immunohistochemical marker of uterine receptivity, the ?v?3 vitronectin.Result(s): Glandular

William R Meyer; Debra B Novotny; Marc A Fritz; Stan A Beyler; Lynda J Wolf; Bruce A Lessey

1999-01-01

271

The Effects of Progesterone on Oocyte Maturation and Embryo Development  

PubMed Central

Oocyte maturation and embryo development are controlled by intra-ovarian factors such as steroid hormones. Progesterone (P4) exists in the follicular fluid that contributes to normal mammalian ovarian function and has several critical functions during embryo development and implantation, including endometrial receptivity, embryonic survival during gestation and transformation of the endometrial stromal cells to decidual cells. It is well known that the physiological effects of P4 during the pre-implantation stages of some mammals embryos are mediated by P4 receptors and their gene expression is determined. The effects of P4 on oocytes and embryo development have been assessed by some investigations, with contradictory results. P4, a dominant steroid in follicular fluid at approximately 18 hours after the luteinizing hormone (LH ) surge may have a critical role in maturation of oocytes at the germinal stage. However, it has been shown that different concentrations of P4 could not improve in vitro maturation rates of germinal vesicles (GV) in cumulus oocyte complexes (COCs) and cumulus denuded oocytes (CDOs). Culture media supplemented with P4 significantly improved mouse embryo development. In addition, an in vivo experimental design has shown high blastocyst survival and implantation rates in P4-treated mice. In this review we explain some of the findings that pertain to the effects of P4 on oocyte maturation and embryo development both in vitro and in vivo.

Salehnia, Mojdeh; Zavareh, Saeed

2013-01-01

272

ROCK inhibitor Y-27632 prevents porcine oocyte maturation.  

PubMed

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

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

2014-07-01

273

Regulation of Greatwall kinase during Xenopus oocyte maturation.  

PubMed

Greatwall kinase has been identified as a key element in M phase initiation and maintenance in Drosophila, Xenopus oocytes/eggs, and mammalian cells. In M phase, Greatwall phosphorylates endosulfine and related proteins that bind to and inhibit protein phosphatase 2A/B55, the principal phosphatase for Cdk-phosphorylated substrates. We show that Greatwall binds active PP2A/B55 in G2 phase oocytes but dissociates from it when progesterone-treated oocytes reach M phase. This dissociation does not require Greatwall kinase activity or phosphorylation at T748 in the presumptive T loop of the kinase. A mutant K71M Greatwall, also known as Scant in Drosophila, induces M phase in the absence of progesterone when expressed in oocytes, despite its reduced stability and elevated degradation by the proteasome. M phase induction by Scant Greatwall requires protein synthesis but is not associated with altered binding or release of PP2A/B55 as compared to wild-type Greatwall. However, in vitro studies with Greatwall proteins purified from interphase cells indicate that Scant, but not wild-type Greatwall, has low but detectable activity against endosulfine. These results demonstrate progesterone-dependent regulation of the PP2A/B55-Greatwall interaction during oocyte maturation and suggest that the cognate Scant Greatwall mutation has sufficient constitutive kinase activity to promote M phase in Xenopus oocytes. PMID:21551066

Yamamoto, Tomomi M; Blake-Hodek, Kristina; Williams, Byron C; Lewellyn, Andrea L; Goldberg, Michael L; Maller, James L

2011-07-01

274

Control of Oocyte Growth and Meiotic Maturation in C. elegans  

PubMed Central

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

Kim, Seongseop; Spike, Caroline; Greenstein, David

2013-01-01

275

Truths and myths of oocyte sensitivity to controlled rate freezing.  

PubMed

The mammalian oocyte is especially sensitive to cryopreservation. Because of its size and physiology, it can easily undergo cell death or sub-lethal damage as a consequence of intracellular ice formation, increase in the concentration of solutes and other undesired effects during the conversion of extracellular water into ice. This has generated the belief that oocyte storage cannot be achieved with the necessary efficiency and safety. However, many concerns raised by oocyte freezing are the result of unproven hypotheses or observations conducted under sometimes inappropriate conditions. For instance, spindle organization can undergo damage under certain freezing conditions but not with other protocols. The controversial suggestion that cryopreservation induces cortical granule discharge and zona pellucida hardening somehow questions the routine use of sperm microinjection. Damage to mouse oocytes caused by solute concentration is well documented but, in the human, there is no solid evidence that modifications of freezing mixtures, to prevent this problem, provide an actual advantage. The hope of developing oocyte cryopreservation as a major IVF option is becoming increasingly realistic, but major efforts are still required to clarify the authentic implications of oocyte cryopreservation at the cellular level and identify freezing conditions compatible with the preservation of viability and developmental ability. PMID:17623530

Coticchio, G; Bonu, M A; Sciajno, R; Sereni, E; Bianchi, V; Borini, A

2007-07-01

276

Functional analysis of oocyte-expressed genes using transgenic models.  

PubMed

An oocyte's journey is highly distinct from the vast majority of cells in the body. As one of the largest and rarest cells, oocytes express unique genes required for the genesis of healthy and competent eggs. The function of only a handful of oocyte-specific genes is beginning to be unraveled. Transgenic mouse models have proven to be extremely valuable in studying the effects of gene deletions on oocytes and surrounding somatic cells. Growth differentiation factor 9 (Gdf9), bone morphogenetic protein 15 (Bmp15), zona pellucida genes (Zp1, Zp2 and Zp3), factor in the germline alpha (Figla), and the c-mos protooncogene (c-mos) are some of the genes preferentially expressed in oocytes which play important roles during folliculogenesis. In order to identify other novel genes preferentially expressed in oocytes, we have utilized subtractive hybridization and in silico subtraction. The combination of these identification approaches, coupled with the use of knockout mice, will lead to many future functional studies of genes uniquely devoted to oogenesis and folliculogenesis. PMID:11988305

Rajkovic, Aleksandar; Matzuk, Martin M

2002-02-22

277

Numerical method to predict projectile penetration  

Microsoft Academic Search

The Simplified Analytical Model of Penetration with Lateral Loading (SAMPLL) computer code developed at Sandia National Laboratories has been modified to allow additional penetration capabilities. The new capabilities include the ability to model penetration by other than cylindrical penetrators (flares, tapers, and boattails) and the ability to calculate penetration\\/perforation of multiple layers of different materials. Additionally, updated soil and rock

L. A. Schoof; F. A. Maestas; C. W. Young

1989-01-01

278

Histopathological changes in juvenile Schistosoma haematobium harboured in hamsters treated with artemether  

Microsoft Academic Search

Histopathological changes in juvenile Schistosoma haematobium, caused by artemether administered to the infected hamsters, were studied. Hamsters were infected with S. haematobium cercariae, and after 28 days, a single dose of artemether (300 mg\\/kg) was administered intragastrically. After 24 h, 72 h and 7 days, groups of two hamsters were sacrificed, and livers were removed, fixed and processed routinely, and

Yang Yuanqing; Xiao Shuhua; Marcel Tanner; Jrg Utzinger; Jacques Chollet; Wu Jiadong; Guo Jian

2001-01-01

279

In vitro fertilizing capacity of sperm from FSH-treated photoinhibited Djungarian hamsters (Phodopus sungorus)  

Microsoft Academic Search

In hypogonadal male Djungarian hamsters FSH alone can induce normal spermatogenesis. However, for the induc- tion of mating behavior, supplementation with testoster- one is necessary. We have here investigated, by in vitro fertilization, whether sperm produced by photoinhibited hamsters treated with FSH alone can fertilize without tes- tosterone. Photoinhibited hypogonadal male Djungarian hamsters were injected daily with human FSH (10

P Niklowitz; A Lerchl; E Nieschlag

1997-01-01

280

Simple or repeated induction of superovulation: a study on ovulation rates and microvessel corrosion casts in ovaries of golden hamsters.  

PubMed

Repeatedly stimulated ovaries are reported to decrease the ovulation rate. One cause among others might be that the microvascular bed has been insufficiently developed. Therefore, 30-day-old golden hamsters were superovulated either once or repeatedly. At the light microscopic level, the ovulation rate in serially sectioned ovaries was indirectly determined by the occurrence of corpora lutea and of abnormal follicle rupture with oocyte release into the cortical stroma (IOR). For the study with scanning electron microscopy (SEM), the microvascular bed of the ovaries was cast with a polyester resin, and the corrosion casts of mature follicles observed. The histological sections of once-stimulated ovaries showed a large number of corpora lutea and IOR follicles. This indicated hyperovulation. In corrosion casts of once-stimulated ovaries, large-sized antral follicles with two layers of a dense capillary meshwork were observed. Capillary sprouts were aligned around the antrum 0 to 12 h after administration of human chorionic gonadotrophin (hCG), and these radiated towards the center of the antrum after 12 to 36 h had elapsed. The ovulation site was recognized at the follicle apex by three similarly sized structures which were either a sinusoid, an oocyte replica, or an opening. Repeatedly stimulated ovaries produced a low number of corpora lutea and almost no IOR follicles. This was judged as hypoovulation. The microvessels of mature follicles were reduced in number and incompletely cast. Widespread resin leakages were conspicuous in the follicle wall 36 h after hCG injection, but the capillary sprouts radiated towards the center of the antrum. No ovulation site was detectable. It is concluded, that capillary sprouts are induced before luteinization. The ovulation site is indicated by particular changes in its microvascular bed. An insufficiently developed microvascular bed may be responsible for hypoovulation in repeatedly stimulated ovaries. PMID:8717322

Lseke, A; Spanel-Borowski, K

1996-01-01

281

Pathogenesis of Modoc Virus (Flaviviridae; Flavivirus) in Persistently Infected Hamsters  

PubMed Central

The long-term persistence of Modoc virus (MODV) infection was investigated in a hamster model. Golden hamsters (Mesocricetus auratus) were infected by subcutaneous inoculation with MODV, in which fatal encephalitis developed in 12.5% (2 of 16). Surviving hamsters shed infectious MODV in their urine during the first five months after infection, and infectious MODV was recovered by co-cultivation of kidney tissue up to eight months after infection. There were no histopathologic changes observed in the kidneys despite detection of viral antigen for 250 days after infection. Mild inflammation and neuronal degeneration in the central nervous system were the primary lesions observed during early infection. These findings confirm previous reports of persistent flavivirus infection in animals and suggest a mechanism for the maintenance of MODV in nature.

Adams, A. Paige; Travassos da Rosa, Amelia P. A.; Nunes, Marcio R.; Xiao, Shu-Yuan; Tesh, Robert B.

2013-01-01

282

The Transcriptome of a Human Polar Body Accurately Reflects Its Sibling Oocyte*  

PubMed Central

Improved methods are needed to reliably and accurately evaluate oocyte quality prior to fertilization and transfer into the woman of human embryos created through in vitro fertilization (IVF). All oocytes that are retrieved and matured in culture are exposed to sperm with little in the way of evaluating the oocyte quality. Furthermore, embryos created through IVF are currently evaluated for developmental potential by morphology, a criterion lacking in quantitation and accuracy. With the recent successes in oocyte vitrification and storage, clear metrics are needed to determine oocyte quality prior to fertilizing. The first polar body (PB) is extruded from the oocyte before fertilization and can be biopsied without damaging the oocyte. Here, we tested the hypothesis that the PB transcriptome is representative of that of the oocyte. Polar body biopsy was performed on metaphase II (MII) oocytes followed by single-cell transcriptome analysis of the oocyte and its sibling PB. Over 12,700 unique mRNAs and miRNAs from the oocyte samples were compared with the 5,431 mRNAs recovered from the sibling PBs (5,256 shared mRNAs or 97%, including miRNAs). The results show that human PBs reflect the oocyte transcript profile and suggests that mRNA detection and quantification through high-throughput quantitative PCR could result in the first molecular diagnostic for gene expression in MII oocytes. This could allow for both oocyte ranking and embryo preferences in IVF applications.

Reich, Adrian; Klatsky, Peter; Carson, Sandra; Wessel, Gary

2011-01-01

283

Projectile penetration into ballistic gelatin.  

PubMed

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

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

2014-01-01

284

Maternal Photoperiodic History Affects Offspring Development in Syrian Hamsters  

PubMed Central

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

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

2009-01-01

285

Impact of oxidative stress in aged mouse oocytes on calcium oscillations at fertilization.  

PubMed

In vivo post-ovulatory aging of oocytes significantly affects the development of oocytes and embryos. Also, oocyte aging alters the regulation of the intracellular calcium concentration, thus affecting Ca(2+) oscillations in fertilized oocytes. Because reactive oxygen species (ROS) are known to significantly perturb Ca(2+) homeostasis mainly through direct effects on the machinery involved in intracellular Ca(2+) storage, we hypothesized that the poor development of aged oocytes that may have been exposed to oxidative stress for a prolonged time might arise from impaired Ca(2+)-oscillation-dependent signaling. The fertilization rates of aged oocytes and of fresh oocytes treated with 100 microM hydrogen peroxide (H(2)O(2)) for 10 min were significantly lower than that of fresh oocytes. Comparing within the fertilized oocytes, blastocyst formation was decreased while embryo fragmentation was increased similarly in the aged and H(2)O(2)-treated fresh oocytes. The frequency of Ca(2+) oscillations was significantly increased whereas the amplitude of individual Ca(2+) transients was lowered in the aged and H(2)O(2)-treated fresh oocytes. The rates of rise and decline in individual Ca(2+) transients were decreased in these oocytes, indicating impaired Ca(2+) handling. When lipid peroxidation was assessed using 4,4-difluoro-5-(4-phenyl-1,3-buttadienyl)-4-bora-3a, 4a-diaza-s-indacene-3-undecanoic acid (C11-BODIPY) in unfertilized oocytes placed in a 5% CO(2) in air atmosphere, the green fluorescence (indicating lipid peroxidation) increased faster in the aged oocytes than in the fresh oocytes. Furthermore, the green fluorescence in the aged oocytes was already approximately 20 times higher than that in the fresh oocytes at the beginning of the measurements. These findings support the idea that Ca(2+) oscillations play a key role in the development of fertilized aged oocytes. PMID:12950101

Takahashi, Toshifumi; Takahashi, Eiji; Igarashi, Hideki; Tezuka, Naohiro; Kurachi, Hirohisa

2003-10-01

286

Determinants of managed care penetration.  

PubMed

This paper examines factors associated with differences in managed care penetration across geographic areas. Two alternative measures of managed care penetration are considered: the percentage of revenue physicians received from managed care contracts and market survey data on enrollments in managed care plans. Results are similar for both types of measures. Our analysis suggests that demographics, labor market characteristics and supply side variables including the level of concentration in hospital markets, hospital occupancy rates and the practice organization patterns of physicians are all important determinants of managed care penetration. PMID:10339250

Dranove, D; Simon, C J; White, W D

1998-12-01

287

Hamster antithrombin III: Purification, characterization and acute phase response  

Microsoft Academic Search

Antithrombin III was purified to homogeneity from hamster plasma by affinity chromatography on heparin-agrose, ion-exchange chromatography on Mono Q and size-exclusion chromatography on TSK G3000SWG column with 50% yield. The molecular mass of hamster antithrombin III was estimated at 62.5 kDa and the absorption coefficient (A280nm1%,1cm) at 6.48 (in 0.1 M sodium phosphate pH 7.0). Several isoforms of the inhibitor

Pawet Mak; Jan J. Enghild; Adam Dubin

1996-01-01

288

Recovery of Leishmania (Viannia) braziliensis from inoculated hamsters.  

PubMed

Leishmania (Viannia) braziliensis has never been isolated from wild animals although it is apparently capable of inducing infections in man, dogs, and donkeys. An analysis of the standard hamster culture system for analyzing infectivity of Leishmania sp. was undertaken. Results indicate that for L. (V.) braziliensis, routine cultivation of aspirates taken from the inoculation sites of 1-mo-infected hamsters should be undertaken. Moreover, in at least 1 of the 3 strains examined, isolation of the parasite was only achieved after 84 days of cultivation. PMID:3379536

Peterson, N E; Costa, M A; Vexenat, J A; Netto, E M; Marsden, P D; Magalhes, A V; Barretto, A C

1988-06-01

289

Discovery and characterization of a new cell-penetrating protein.  

PubMed

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

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

2013-12-20

290

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

NASA Astrophysics Data System (ADS)

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.

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

291

Skin Penetration Mechanisms of Helminths.  

National Technical Information Service (NTIS)

The structure and physiology of human skin are described and related to the following penetration mechanisms of the Schistosoma mansoni cercaria: Brief exploration of the sebum-covered skin surface for entry sites. Attempted entry at irregularities associ...

M. A. Stirwalt

1966-01-01

292

Cement penetration after patella venting.  

PubMed

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

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

2009-01-01

293

Penetrant-Indication-Measuring Compass  

NASA Technical Reports Server (NTRS)

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.

Schaefer, Lloyd

1991-01-01

294

mRNA from NCB-20 cells encodes the N-methyl-D-aspartate/phencyclidine receptor: a Xenopus oocyte expression study.  

PubMed Central

The mouse neuroblastoma--Chinese hamster brain hybrid cell line NCB-20 is the only clonal cell line in which binding studies indicate the presence of phencyclidine (PCP) receptors. We report here that Xenopus oocytes injected with NCB-20 cell poly(A)+ RNA express N-methyl-D-aspartate (NMDA)-activated channels and that these channels include the PCP receptor site. In injected oocytes, NMDA application evoked a partially desensitizing inward current that was potentiated by glycine, blocked by the competitive antagonist D-2-amino-5-phosphonovaleric acid, blocked by Mg2+ and by Zn2+, and blocked in a use-dependent manner by the PCP receptor ligands PCP and MK-801. There was little or no response to kainate or quisqualate (agonists of the other excitatory amino acid receptors), to gamma-aminobutyric acid (an inhibitory transmitter), or to glycine (an inhibitory transmitter as well as an allosteric potentiator of NMDA channels). Thus, NMDA/PCP receptors expressed from NCB-20 cell mRNA exhibit properties similar to those of the neuronal receptors. The absence of expression of other excitatory amino acid receptors in this system makes it particularly useful for study of NMDA-evoked responses without interference from responses mediated by other receptors. Moreover, NCB-20 mRNA may be an appropriate starting material for cloning the cDNA(s) encoding the NMDA/PCP-receptor complex.

Lerma, J; Kushner, L; Spray, D C; Bennett, M V; Zukin, R S

1989-01-01

295

Potential applications of sheep oocytes as affected by vitrification and in vitro aging.  

PubMed

The present study was carried out to investigate how the interactions between aging, vitrification and post-warming interval affect the credibility of sheep MII-oocytes for in vitro fertilization (IVF), intracytoplasmic injection (ICSI), and parthenogenetic activation (PA). According to our results, aged oocytes had significantly higher rates of chromosome and spindle abnormalities compared to young oocytes. However after vitrification-warming, the total rates of these abnormalities were not significantly different between aged and young oocytes. Unvitrified-aged, and vitrified young and aged oocytes had comparable ultrastructural characteristics, whereas they were completely dissimilar in compared with unvitrified-young oocytes. Although mRNA abundance was reduced during vitrification-warming in both aged and young oocytes, the post-warming interval could improve the relative mRNA abundance. Aged oocytes had lower capacity for IVF and ICSI in compared with young oocytes, but had similar pattern for PA process. The vitrification process decreased developmental competence of both aged and young oocytes in compared with young ones, particularly when warmed oocytes were rested for 2 h before IVF, ICSI and PA. The results of the present study showed that in vitro aged oocytes had higher capacity to be used for parthenogenetic studies rather than IVF and ICSI. Furthermore, it was shown that vitrified oocytes had a time-dependent decline in quality and developmental potential. Notably, the speed of this decline was higher in vitrified-young oocytes, indicating that the vitrified oocytes do not require to be rested post warming. Conclusively, the results of this study can be useful in preserving in vitro aged oocytes to provide a valuable and easy access source of oocytes for research purposed studies. PMID:22444551

Hosseini, S M; Asgari, V; Ostadhosseini, S; Hajian, M; Piryaei, A; Najarasl, M; Nasr-Esfahani, M H

2012-06-01

296

Evidence for dysregulation of genome-wide recombination in oocytes with nondisjoined chromosomes 21.  

PubMed

In oocytes with nondisjoined chromosomes 21 due to a meiosis I (MI) error, recombination is significantly reduced along chromosome 21; several lines of evidence indicate that this contributes to the nondisjunction event. A pilot study found evidence that these oocytes also have reduced recombination genome-wide when compared with controls. This suggests that factors that act globally may be contributing to the reduced recombination on chromosome 21, and hence, the nondisjunction event. To identify the source of these factors, we examined two levels of recombination count regulation in oocytes: (i) regulation at the maternal level that leads to correlation in genome-wide recombination across her oocytes and (ii) regulation at the oocyte level that leads to correlation in recombination count among the chromosomes of an oocyte. We sought to determine whether either of these properties was altered in oocytes with an MI error. As it relates to maternal regulation, we found that both oocytes with an MI error (N = 94) and their siblings (N = 64) had reduced recombination when compared with controls (N = 2723). At the oocyte level, we found that the correlation in recombination count among the chromosomes of an oocyte is reduced in oocytes with MI errors compared with that of their siblings or controls. These results suggest that regulation at the maternal level predisposes MI error oocytes to reduced levels of recombination, but additional oocyte-specific dysregulation contributes to the nondisjunction event. PMID:24014426

Middlebrooks, Candace D; Mukhopadhyay, Nandita; Tinker, Stuart W; Allen, Emily Graves; Bean, Lora J H; Begum, Ferdouse; Chowdhury, Reshmi; Cheung, Vivian; Doheny, Kimberly; Adams, Marcia; Feingold, Eleanor; Sherman, Stephanie L

2014-01-15

297

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

PubMed Central

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

Redi, Carlo Alberto; Zuccotti, Maurizio

2014-01-01

298

Metabolic fuel homeostasis in golden hamsters: effects of fasting, refeeding, glucose, and insulin.  

PubMed

Experiments were conducted to investigate possible metabolic correlates of the unusual ingestive behavior of hamsters after food deprivation. A hypothesis of metabolic refractoriness predicts that hamsters, unlike rats, should not show changes in plasma metabolic fuels, adipose tissue, or liver after fasting and subsequent refeeding. This hypothesis was discredited by findings that fasted hamsters, like rats, have increased plasma ketones and free fatty acids and decreased liver glycogen. On refeeding, hamsters showed rapid reversal of these changes, with supranormal glycogen content and apparent fatty acid synthesis in liver. Additional studies examined the metabolic responses of hamsters and rats to exogenous insulin or glucose administration. Incorporation of 3H2O into liver fatty acids was greatly elevated in rats by both insulin and glucose, but in hamsters only insulin was effective. Some of these metabolic differences may help our understanding of the unusual refractoriness of hamster food intake to various stimuli. PMID:6377929

Rowland, N

1984-07-01

299

Evaluation of sperm subpopulation structure in relation to invitro sperm-oocyte interaction of frozen-thawed semen from Holstein bulls.  

PubMed

The present study examined the relationship between the relative amount of high motile sperm and sperm-oocyte interactions obtained from Holstein bull ejaculates. Post-thaw sperm motility was analyzed using a computer-assisted sperm analyzer system and evaluated to determine the sperm motility subpopulations. Adhesion and penetration of zona pellucida (ZP) and pronucleus formation using post-thawed samples (15 ejaculates form 5 different bulls) with different percentages of sperm in the subpopulation with the fastest and most progressive subpopulation (subpopulation 4 [SP4]) were analyzed. The correlation between the proportion of sperm in SP4 and the number of spermatozoa bound to the zona pellucida (ZBA), the penetration rate, and the rate of pronucleus formation were calculated. A significant (P < 0.05) and positive correlation was found between the number of spermatozoa bound to the zona pellucida, the penetration rate, and the rate of pronucleus formation with the proportion of sperm in SP4 (r = 0.79, r = 0.66, and r = 0.63, respectively). Our results suggest that this specific high motile and progressive subpopulation is positively and significantly correlated with the ability of a thawed bull semen sample to interact properly with the oocyte and its extracellular vestments. These findings emphasize the relevance of analyzing semen subpopulation composition to predict bull sperm fertilizing ability and to select Holstein bulls for breeding purposes. PMID:24581584

Ferraz, M A M M; Morat, R; Yeste, M; Arcarons, N; Pena, A I; Tamargo, C; Hidalgo, C O; Muio, R; Mogas, T

2014-05-01

300

Hamster challenge potency assay for evaluation of Mycoplasma pneumoniae vaccines.  

PubMed Central

A hamster immunization challenge assay described in the accompanying paper (M. F. Barile, D. K. F. Chandler, H. Yoshida, M. W. Grabowski, R. Harasawa, and S. Razin, Infect. Immun. 56:2443-2449, 1988) was used to examine protection against Mycoplasma pneumoniae disease by passive immunization and to evaluate the protective potency of a Formalin-inactivated whole-cell and a cell extract M. pneumoniae vaccine. Passive immunization with a globulin fraction of hyperimmune mule antiserum to M. pneumoniae provided hamsters some protection against the challenge. When hamsters were actively immunized, a single dose of Formalin-inactivated vaccine provided only minimal protection, whereas multiple doses of this vaccine, particularly when combined with adjuvant, provided good protection. A single dose of the cell extract vaccine did not protect animals, but two doses caused a marked reduction of disease when a priming dose was given intraperitoneally, followed by a booster dose intratracheally. The correlation between the level of metabolism inhibition antibodies to M. pneumoniae in the sera of vaccinated hamsters and the degree of protection as measured by reduction of lung pathological scores and colonization was poor, indicating that seroconversion rates for metabolism inhibition antibodies are not by themselves adequate to measure the potency of M. pneumoniae vaccines.

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

1988-01-01

301

Banding and chromatid separation in Chinese hamster chromosomes  

Microsoft Academic Search

IN general, a chromosome exhibiting its banding pattern does not permit any internal helix to be seen, and vice versa. During studies with chromosomes of Chinese hamster cells, cultivated in vitro, it was possible to correlate G banding and spiral structure with the spatial relation between sister chromatids. G banding of human chromosomes was first described by Sumner et al.1

H. Hatami-Monazah

1974-01-01

302

Biological Effect of Focal alpha Radiation on the Hamster Lung.  

National Technical Information Service (NTIS)

Monodispersed 10- mu m diameter ZrO sub 2 ceramic microspheres, containing varying amounts of exp 239 PuO sub 2 or exp 238 PuO sub 2 , were injected into the jugular vein of 100-day-old Syrian hamsters. These biologically inert microspheres lodged subsequ...

D. M. Smith E. C. Anderson J. R. Prine L. M. Holland C. R. Richmond

1975-01-01

303

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 ...

304

Behavior evoked by electrical stimulation of the hamster superior colliculus  

Microsoft Academic Search

Syrian golden hamsters were implanted with fixed or moveable stimulating electrodes aimed at the superior colliculus (SC). Behavior was observed in response to trains of 0.1 ms pulses at 200 Hz while the animals were moving freely in an open arena or in their home cages. At threshold stimulating currents, the responses consisted almost entirely of freezing or contraversive turning,

D. P. M. Northmore; E. S. Levine; G. E. Schneider

1988-01-01

305

Parameters of Mycoplasma pneumoniae infection in Syrian hamsters.  

PubMed Central

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

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

1988-01-01

306

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...

307

Natural Resistance of Hamster Cells to 8-Azaguanine.  

National Technical Information Service (NTIS)

In studies designed to introduce mutations into selected animal cell lines in culture, cell lines derived from the Syrian hamster were found to be naturally resistant to 8-azaguanine. Resistance to the drug was measured by plating colony-forming units at ...

A. Richter

1965-01-01

308

Photoperiodic Regulation of Compensatory Testicular Hypertrophy in Hamsters1  

Microsoft Academic Search

In mammals, removal of one testis results in compensatory testicular hypertrophy (CTH) of the remaining gonad. Although CTH is ubiquitous among juveniles of many species, laboratory rats, laboratory mice, and humans unilaterally castrated in adulthood fail to display CTH. We documented CTH in pre- and postpubertally hemi-castrated Syrian and Siberian hamsters and tested whether day length affects CTH in juvenile

Matthew J. Paul; Jin Ho Park; Teresa H. Horton; Maria I. Alvarez; Morgan K. Burke; Irving Zucker

309

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...

310

Photoperiod and stress affect wound healing in Siberian hamsters  

Microsoft Academic Search

Changes in day length alter several indices of immune function in Siberian hamsters. These experiments tested the hypothesis that photoperiodic changes in immune function are integrated at an organismal level as reflected by the ability to heal a cutaneous wound. Given the well-documented effects of psychological stressors on immune function, we also tested the hypothesis that photoperiod modulates the effects

Steven G. Kinsey; Brian J. Prendergast; Randy J. Nelson

2003-01-01

311

BIODYNAMIC STUDIES OF HAMSTER FLANK ORGAN GROWTH: HORMONAL INFLUENCES  

Microsoft Academic Search

The biodynamic response of flank organs of male and female hamsters to androgenic stimulation has been studied by autoradiographic and electron microscopic techniques, as well as by routine histology and gross observation. Intraperitoneal administration of 2.5 mg of testosterone on alternate days resulted in bilateral increase in palpable bulk, and pigmentation of flank organs of females, males, and castrated males.

Phillip Frost; Joseph L. Giegel; Gerald D. Weinstein; Edward C. Gomez

1973-01-01

312

[Changes in amino acid metabolism at the onset of colchicine resistance in Dzungarian hamster fibroblast cell culture].  

PubMed

We have studied amino acid up-take from incubation media by colchicine-resistant and colchicine-sensitive Djungarian hamster fibroblast cells. It has been shown that arginine and asparagine amino acid contents in the medium are different for colchicine-sensitive and colchicine-resistant cells. Amino acids concentration is not reduced in the medium after incubation of resistant cells, while it is decreased after incubation of sensitive cells. We can suggest that penetration of low-weight sources of nitrogen into sensitive cells is hampered. It can also be suggested that protein macromolecules are the main source of nitrogen for these cells. The protein up-take levels from the incubation medium, as assessed by the ammonia and amino acid contents of cell counts don't exceed 5% of their initial concentrations in the medium. PMID:2597768

Moroz, L V; Donenko, F V; Borovkova, N B; Kushelev, A R

1989-10-01

313

Age-Related Decrease of Meiotic Cohesins in Human Oocytes  

PubMed Central

Aneuploidy in fetal chromosomes is one of the causes of pregnancy loss and of congenital birth defects. It is known that the frequency of oocyte aneuploidy increases with the human maternal age. Recent data have highlighted the contribution of cohesin complexes in the correct segregation of meiotic chromosomes. In mammalian oocytes, cohesion is established during the fetal stages and meiosis-specific cohesin subunits are not replenished after birth, raising the possibility that the long meiotic arrest of oocytes facilitates a deterioration of cohesion that leads to age-related increases in aneuploidy. We here examined the cohesin levels in dictyate oocytes from different age groups of humans and mice by immunofluorescence analyses of ovarian sections. The meiosis-specific cohesin subunits, REC8 and SMC1B, were found to be decreased in women aged 40 and over compared with those aged around 20 years (P<0.01). Age-related decreases in meiotic cohesins were also evident in mice. Interestingly, SMC1A, the mitotic counterpart of SMC1B, was substantially detectable in human oocytes, but little expressed in mice. Further, the amount of mitotic cohesins of mice slightly increased with age. These results suggest that, mitotic and meiotic cohesins may operate in a coordinated way to maintain cohesions over a sustained period in humans and that age-related decreases in meiotic cohesin subunits impair sister chromatid cohesion leading to increased segregation errors.

Tsutsumi, Makiko; Fujiwara, Reiko; Nishizawa, Haruki; Ito, Mayuko; Kogo, Hiroshi; Inagaki, Hidehito; Ohye, Tamae; Kato, Takema; Fujii, Takuma; Kurahashi, Hiroki

2014-01-01

314

Molecular control of oocyte meiotic arrest and resumption.  

PubMed

Mammalian oocytes within Graafian follicles are arrested at prophase I by factors from surrounding follicle cells, and resume meiosis after an LH surge from the pituitary. The maintenance of meiotic arrest requires high levels of cAMP, resulting from G-protein-coupled receptor (GPR) 3 and/or GPR12 activation of adenylyl cyclase within the oocyte. Recent studies indicate that natriuretic peptide precursor C (NPPC), acting via its cognate receptor NPR2, increases cGMP levels in granulosa cells; the cGMP then diffuses into oocytes and inhibits phosphodiesterase 3A activity and cAMP hydrolysis. Meiotic resumption is induced by LH via the generation of epidermal growth factor (EGF)-like growth factors in mural granulosa cells that activate EGF receptors in cumulus cells. However, the exact mechanisms underlying the actions of these growth factors on oocyte maturation are unclear. Herein we summarise the regulatory functions of NPPC and NPR2 in maintaining oocyte meiotic arrest and discuss the possibility that LH could stimulate meiotic resumption by decreasing NPPC content and NPR2 activity. PMID:23217677

Liu, Lei; Kong, Nana; Xia, Guoliang; Zhang, Meijia

2013-01-01

315

Use of fetuin before and during vitrification of bovine oocytes.  

PubMed

After vitrification of oocytes, fertilization rates and subsequent development are unsatisfactory, possibly due in part to zona hardening. Foetal calf serum (FCS) can prevent zona hardening because of its fetuin content, but FCS composition varies among batches, and may contain viruses. In this study, we therefore compared media supplemented with different sources of macromolecules, 2% bovine serum albumin (BSA), 2% BSA + 1 mg/ml fetuin and 20% FCS, for handling oocytes for 10-30 min prior to vitrification. None of the treatments resulted in developmental rates comparable with the non-vitrified controls, but FCS inclusion in pre-vitrification handling medium resulted in higher blastocyst production per oocyte (p < 0.05) (10.8%) on day 9 of culture than BSA (5.3%) or BSA + fetuin (6.4%). Blastocysts developing from oocytes from all vitrification treatments were somewhat retarded relative to those developed from non-vitrified oocytes. We also tested the use of fetuin during vitrification as well as two different exposure times with cryoprotectants, 180 and 30 s. There was no significant effect of fetuin or exposure time on rates of subsequent blastocyst production. PMID:18069945

Horvath, G; Seidel, G E

2008-06-01

316

Recombination and DNA Replication in the DROSOPHILA MELANOGASTER Oocyte  

PubMed Central

A method is described that permits the recovery of a well-synchronized population of oocytes. Utilizing this pupal system, the heat-responsive period for increasing crossing-over in the Drosophila genome has been defined for the X chromosome and a portion of chromosome 2. The response is initiated close to the time of oocyte formation (premeiotic interphase) and is terminated after ?36 hr. During the 36-hr period different regions show characteristic responses, which vary in degree, in duration, and in initiation and termination points, so as to generate the beginning of a thermal recombination map for the Drosophila genome. Centromere regions exhibit the greatest increases in crossing-over for their respective chromosomes but are distinctly asynchronous in time; interstitial regions respond the least. Correlated autoradiographic studies have localized DNA replication in the oocyte to a ?24-hr period, which also begins close to oocyte formation (premeiotic interphase); late labeling in restricted regions, undetectable with the present method, could extend the period, as could prolonged synthesis in the oocyte. The results demonstrate that DNA replication and the heat-sensitive period for enhancement of crossing-over are coincident processes over most and possibly all of their length.

Grell, Rhoda F.

1973-01-01

317

Proteomic analysis of mammalian gametes and sperm-oocyte interactions.  

PubMed

Proteomic analysis occupies an increasingly important place in gamete and embryo biology as an independent tool of discovery and as a means of follow-up to transcriptional profiling. Proteomics have been and will be increasingly helpful in many areas of reproductive biology, including applied science and technology development. Areas likely to be impacted most rapidly by proteomic knowledge include fertility evaluation in male farm animals, male infertility diagnostics in humans, assessment and optimization of oocyte and embryo culture protocols, selection of fittest oocytes for assisted fertilization and selection of most competent embryos for embryo transfer. Oocyte proteomics will help us understand the process of oogenesis and oocyte maturation, and to discover non-invasive markers of oocyte quality. Sperm proteomics correlate with normal sperm structure and function and can be applied to discover novel biomarkers of farm animal fertility and diagnostic markers of human male infertility. Putative receptors participating in fertilization, as well as proteins acquired onto sperm surface from epididymal fluid and seminal plasma, have been discovered by proteomic analysis. An added level of information is provided by advanced proteomic approaches, capable of identifying posttranslational modifications such as phosphorylation, glycosylation and ubiquitination which play important functions in gametogenesis, fertilization and embryo development. By no means exhaustive, the present paper reviews some of the most interesting proteomic studies of mammalian gametes and embryos published in the last decade. PMID:19848273

Sutovsky, P

2009-01-01

318

Drosha protein levels are translationally regulated during Xenopus oocyte maturation.  

PubMed

MicroRNAs (miRNAs) are ?21-nucleotide-long, single-stranded noncoding RNAs that regulate gene expression. Biogenesis of miRNAs is mediated by the two RNase III-like enzymes, Drosha and Dicer. Here we study miRNA biogenesis during maturation of Xenopus oocytes to eggs using microinjection of pri-miRNAs. We show that processing of exogenous and endogenous primary miRNAs (pri-miRNAs) is strongly enhanced upon maturation of oocytes to eggs. Overexpression of cloned Xenopus Drosha in oocytes, however, boosts pri-miRNA processing dramatically, indicating that Drosha is a rate-limiting factor in Xenopus oocytes. This developmental regulation of Drosha is controlled by poly(A) length addition to the Drosha mRNA, which boosts translation upon transition from oocytes to eggs. Processing of pri-miRNAs by Drosha and Dicer has been shown to be affected by adenosine-to-inosine deamination-type RNA editing. Using activated Xenopus eggs for microinjection experiments, we demonstrate that RNA editing can reduce pri-miRNA processing in vivo. This processing block is determined by the structural but not sequence changes introduced by RNA editing. PMID:24829383

Muggenhumer, Dominik; Vesely, Cornelia; Nimpf, Simon; Tian, Nan; Yongfeng, Jin; Jantsch, Michael F

2014-07-01

319

The GABA A receptor subunits heterologously expressed in Xenopus oocytes.  

PubMed

The ?-aminobutyric acid (GABA) A receptor is composed of a variety of subunits and combinations and shows a characteristic distribution in the CNS. To date, 20 subunits of the GABA A receptor have been cloned: ?1-6, ?1-4, ?1-3, ?, ?, ? , ?, and ?1-3. Oocyte of Xenopus laevis is one of the most frequently used heterologous expression systems, which are used to design and analyze specific combinations of GABA A receptor subunits. In oocytes, a certain GABA A receptor function is studied only by comparing the amplitude of the response to GABA and other drugs by physiological and pharmacological methods. According to the studies on Xenopus laevis oocytes, the ?1?2?2S receptor combination is mostly used. The ?1-containing receptors mediate sedative and anticonvulsant acts. The results of studies on oocytes show that PKA, NKCC1, P2X3 receptors, and GABA A receptor-associated protein, etc., are existing systems that show different reactivity to the GABA A receptors. The GABA A receptor subunits contain distinct binding sites for BZDs, neurosteroids, general anesthetics, etc., which are responsible for the numerous functions of the GABA A receptor. A variety of other drugs, such as topiramate, TG41, (+)- and (-)-borneol, apigenin, and 6-methylflavone could also have modulatory effects on the GABA A receptors. Some of the different models and hypotheses on GABA A receptor structure and function have been achieved by using the two-electrode voltage clamp method in oocytes. PMID:23373649

Abdullah, Jafri Malin; Zhang, Jingli

2013-04-01

320

FISH Analysis of Six Chromosomes in Unfertilized Human Oocytes After Polar Body Removal  

Microsoft Academic Search

Purpose: To develop an improved technique for estimatingchromosomal abnormalities in human oocytes byfluorescence in situ hybridization (FISH) and to correlate theposition of single chromatids with the chromosomal status ofthe oocytes.

Elena Martini; Sean P. Flaherty; Nicholas J. Swann; Colin D. Matthews; Frans C. S. Ramaekers; Joep P. M. Geraedts

2000-01-01

321

High-throughput optofluidic system for the laser microsurgery of oocytes  

NASA Astrophysics Data System (ADS)

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.

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

2012-01-01

322

In silico identification and molecular characterization of genes predominantly expressed in the fish oocyte  

Microsoft Academic Search

BACKGROUND: In fish, molecular mechanisms that control follicle-enclosed oocyte progression throughout oogenesis and oocyte developmental competence acquisition remain poorly understood. Existing data in mammals have indicated that the so called \\

Julien Bobe; Thaovi Nguyen; Sophie Mah; Philippe Monget

2008-01-01

323

Measurement of ATP in single oocytes: impact of maturation and cumulus cells on levels and consumption.  

PubMed

Mitochondria provide the primary source of ATP in the oocyte and early embryo and mitochondrial dysfunction and deficit of mitochondria-derived ATP has been linked to suboptimal developmental competence. We have undertaken a study of ATP in the maturing mouse oocyte using a novel recombinant FRET based probe, AT1.03. We show that AT1.03 can be successfully used to monitor cytosolic ATP levels in single live oocytes over extended time periods. We find that ATP levels undergo dynamic changes associated with specific maturational events and that oocytes display altered rates of ATP consumption at different stages of maturation. Cumulus enclosed oocytes have a higher ATP level during maturation than denuded oocytes and this can be abolished by inhibition of gap junctional communication between the oocyte and cumulus cells. Our work uses a new approach to shed light on regulation of ATP levels and ATP consumption during oocyte maturation. PMID:24002908

Dalton, Caroline M; Szabadkai, Gyorgy; Carroll, John

2014-03-01

324

Cytoplasmic Microtubular Dynamics and Chromatin Organization during Mammalian Oogenesis and Oocyte Maturation.  

National Technical Information Service (NTIS)

A chronological series of coordinated alterations in oocyte chromosome and microtubule disposition occur during oogenesis and oocyte maturation in the mammal. Timely transitions in meiotic spindle and cytoplasmic microtubules, due to modifications in both...

D. F. Albertini

1992-01-01

325

TGF-? and Insulin Signaling Regulate Reproductive Aging via Oocyte and Germline Quality Maintenance  

PubMed Central

SUMMARY Reproductive cessation is perhaps the earliest aging phenotypes humans experience. Similarly, C. elegans reproduction ceases in mid-adulthood. Although somatic aging has been studied in both worms and humans, mechanisms regulating reproductive aging are not yet understood. Here we show that TGF-? Sma/Mab and Insulin/IGF-1 signaling regulate C. elegans reproductive aging by modulating multiple aspects of the reproductive process, including embryo integrity, oocyte fertilizability, chromosome segregation fidelity, DNA damage resistance, and oocyte and germline morphology. TGF-? activity regulates reproductive span and germline/oocyte quality non-cell-autonomously, and is temporally and transcriptionally separable from its regulation of growth. Chromosome segregation, cell cycle, and DNA damage response genes are upregulated in TGF-? mutant oocytes, decline in aged mammalian oocytes, and are critical for oocyte quality maintenance. Our data suggest that C. elegans and humans share many aspects of reproductive aging, including the correlation between reproductive aging and declining oocyte quality, and mechanisms determining oocyte quality.

Luo, Shijing; Kleemann, Gunnar A.; Ashraf, Jasmine M.; Shaw, Wendy M.; Murphy, Coleen T.

2010-01-01

326

Instructing an Embryonic Stem Cell-Derived Oocyte Fate: Lessons from Endogenous Oogenesis  

PubMed Central

Female reproductive potential is limited in the majority of species due to oocyte depletion. Because functional human oocytes are restricted in number and accessibility, a robust system to differentiate oocytes from stem cells would enable a thorough investigation of the genetic, epigenetic, and environmental factors affecting human oocyte development. Also, the differentiation of functional oocytes from stem cells may permit the success of human somatic cell nuclear transfer for reprogramming studies and for the production of patient-specific embryonic stem cells (ESCs). Thus, ESC-derived oocytes could ultimately help to restore fertility in women. Here, we review endogenous and ESC-derived oocyte development, and we discuss the potential and challenges for differentiating functional oocytes from ESCs.

Nicholas, Cory R.; Chavez, Shawn L.; Baker, Valerie L.; Reijo Pera, Renee A.

2009-01-01

327

Anti-Mllerian hormone (AMH), inhibin-?, growth differentiation factor 9 (GDF9), and bone morphogenic protein-15 (BMP15) mRNA and protein are influenced by photoperiod-induced ovarian regression and recrudescence in Siberian hamster ovaries.  

PubMed

Exposure of Siberian hamsters to short photoperiod (SD) inhibits ovarian function, including folliculogenesis, whereas function is restored with their transfer to long photoperiods (LD). To investigate the mechanism of photo-stimulated recrudescence, we assessed key folliculogenic factors-anti-Mllerian hormone (AMH), inhibin-?, growth differentiation factor-9 (GDF9), and bone morphogenic protein-15 (BMP15)-across the estrus cycle and in photo-regressed and recrudescing ovaries. Adult hamsters were exposed to either LD or SD for 14 weeks, which respectively represent functional and regressed ovaries. Select regressed hamsters were transferred back to LD for 2 (post-transfer week 2; PTw2) or 8 weeks (PTw8). Ovaries were collected and fixed in formalin for immunohistochemistry or frozen in liquid nitrogen for real-time PCR. AMH, inhibin-?, GDF9, and BMP15 mRNA and protein were detected in all stages of the estrus cycle. Fourteen weeks of SD exposure increased (P?hamsters to stimulatory long photoperiod for 8 weeks returned AMH and GDF9 mRNA levels to LD-treated levels, and further increased mRNA levels for inhibin-? and BMP15. Immunostaining for AMH, inhibin-?, GDF9, and BMP15 proteins was most intense in preantral/antral follicles and oocytes. The overall immunostaining extent for AMH and inhibin-? generally mirrored the mRNA data, though no changes were observed for GDF9 or BMP15 immunostaining. Shifts in mRNA and protein levels across photoperiod conditions suggest possible syncretic roles for these folliculogenic factors in photo-stimulated recrudescence via potential regulation of follicle recruitment, preservation, and development. PMID:23877969

Shahed, Asha; Young, Kelly A

2013-11-01

328

Pendrin Function and Regulation in Xenopus Oocytes  

PubMed Central

SLC26A4/PDS mutations cause Pendred Syndrome and non-syndromic deafness. but some aspects of function and regulation of the SLC26A4 polypeptide gene product, pendrin, remain controversial or incompletely understood. We have therefore extended the functional analysis of wildtype and mutant pendrin in Xenopus oocytes, with studies of isotopic flux, electrophysiology, and protein localization. Pendrin mediated electroneutral, pH-insensitive, DIDS-insensitive anion exchange, with extracellular K(1/2) (in mM) of 1.9 (Cl?), 1.8 (I?), and 0.9 (Br?). The unusual phenotype of Pendred Syndrome mutation E303Q (loss-of-function with normal surface expression) prompted systematic mutagenesis at position 303. Only mutant E303K exhibited loss-of-function unrescued by forced overexpression. Mutant E303C was insensitive to charge modification by methanethiosulfonates. The corresponding mutants SLC26A2 E336Q, SLC26A3 E293Q, and SLC26A6 E298Q exhibited similar loss-of-function phenotypes, with wildtype surface expression also documented for SLC26A2 E336Q. The strong inhibition of wildtype SLC26A2, SLC26A3, and SLC26A6 by phorbol ester contrasts with its modest inhibition of pendrin. Phorbol ester inhibition of SLC26A2, SLC26A3, and SLC26A6 was blocked by coexpressed kinase-dead PKC? but was without effect on pendrin. Mutation of SLC26A2 serine residues conserved in PKC? -sensitive SLC26 proteins but absent from pendrin failed to reduce PKC? sensitivity of SLC26A2 (190).

Reimold, Fabian R.; Heneghan, John F.; Stewart, Andrew K.; Zelikovic, Israel; Vandorpe, David H.; Shmukler, Boris E.; Alper, Seth L.

2011-01-01

329

Pendrin function and regulation in Xenopus oocytes.  

PubMed

SLC26A4/PDS mutations cause Pendred Syndrome and non-syndromic deafness. but some aspects of function and regulation of the SLC26A4 polypeptide gene product, pendrin, remain controversial or incompletely understood. We have therefore extended the functional analysis of wildtype and mutant pendrin in Xenopus oocytes, with studies of isotopic flux, electrophysiology, and protein localization. Pendrin mediated electroneutral, pH-insensitive, DIDS-insensitive anion exchange, with extracellular K((1/2)) (in mM) of 1.9 (Cl(-)), 1.8 (I(-)), and 0.9 (Br(-)). The unusual phenotype of Pendred Syndrome mutation E303Q (loss-of-function with normal surface expression) prompted systematic mutagenesis at position 303. Only mutant E303K exhibited loss-of-function unrescued by forced overexpression. Mutant E303C was insensitive to charge modification by methanethiosulfonates. The corresponding mutants SLC26A2 E336Q, SLC26A3 E293Q, and SLC26A6 E298Q exhibited similar loss-of-function phenotypes, with wildtype surface expression also documented for SLC26A2 E336Q. The strong inhibition of wildtype SLC26A2, SLC26A3, and SLC26A6 by phorbol ester contrasts with its modest inhibition of pendrin. Phorbol ester inhibition of SLC26A2, SLC26A3, and SLC26A6 was blocked by coexpressed kinase-dead PKC? but was without effect on pendrin. Mutation of SLC26A2 serine residues conserved in PKC? -sensitive SLC26 proteins but absent from pendrin failed to reduce PKC? sensitivity of SLC26A2 (190). PMID:22116357

Reimold, Fabian R; Heneghan, John F; Stewart, Andrew K; Zelikovic, Israel; Vandorpe, David H; Shmukler, Boris E; Alper, Seth L

2011-01-01

330

Birth of a healthy female after intracytoplasmic sperm injection of cryopreserved human oocytes  

Microsoft Academic Search

Objective: To describe the first birth achieved after intracytoplasmic sperm injection (ICSI) of cryopreserved human oocytes.Design: Case report.Setting: University of Bologna Hospital, Department of Obstetrics and Gynecology, Reproductive Endocrinology Unit, IVF and Infertility Center.Patient(s): One patient undergoing IVF.Intervention(s): Transvaginal ultrasound-guided oocyte retrieval followed by oocyte freezing. Artificial preparation of the endometrium with E2 and P, oocyte thawing, and ICSI.Result(s): Four

Eleonora Porcu; Raffaella Fabbri; Renato Seracchioli; Patrizia M. Ciotti; Otello Magrini; Carlo Flamigni

1997-01-01

331

Birth after cryopreservation of immature oocytes with subsequent in vitro maturation  

Microsoft Academic Search

Objective: To establish the clinical feasibility of using cryostored germinal vesicle oocytes for IVF and ET.Design: Case report.Setting: Private infertility clinic.Patient(s): A 28-year-old woman with tubal infertility undergoing IVF therapy.Intervention(s): Oocytes collected after ovarian stimulation were frozen without insemination or were inseminated, fertilized, and frozen as cleavage stage embryos. No fresh oocyte or embryo transfer was undertaken. All oocytes were

Michael J Tucker; Graham Wright; Paula C Morton; Joe B Massey

1998-01-01

332

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

PubMed

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 ?m 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

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

333

Cryopreservation of mature human oocytes by vitrification with ethylene glycol in straws  

Microsoft Academic Search

Objective: To examine the effect of vitrification with ethylene glycol (EG) for mature human oocytes in straws.Design: Prospective, randomized, in vitro experiments.Setting: Reproductive unit of a university hospital.Patient(s): Immature oocytes from 110 patients undergoing intracytoplasmic sperm injection (ICSI).Intervention(s): The immature oocytes were incubated to reach metaphase II (MII). The MII oocytes were treated with EG-based cryoprotectants and vitrified in straws.

Shee-Uan Chen; Yih-Ron Lien; Kuang-Han Chao; Hsin-Fen Lu; Hong-Nerng Ho; Yu-Shih Yang

2000-01-01

334

Calcium-Dependent Transmitter Secretion Reconstituted in Xenopus Oocytes: Requirement for Synaptophysin  

Microsoft Academic Search

Calcium-dependent glutamate secretion was reconstituted in Xenopus oocytes by injecting the oocyte with total rat cerebellar messenger RNA (mRNA). Co-injection of total mRNA with antisense oligonucleotides to synaptophysin message decreased the expression of synaptophysin in the oocyte and reduced the calcium-dependent secretion. A similar effect on secretion was observed for oocytes injected with total mRNA together with an antibody to

Janet Alder; Bai Lu; Flavia Valtorta; Paul Greengard; Mu-Ming Poo

1992-01-01

335

Double-Plate Penetration Equations  

NASA Technical Reports Server (NTRS)

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.

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

2000-01-01

336

Simulation of laser penetration efficiency  

NASA Astrophysics Data System (ADS)

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

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

2013-09-01

337

Does ovarian hyperstimulation syndrome affect the quality of oocytes?  

PubMed

Two wives of a Muslim with severe male factor infertility had simultaneous intracytoplasmic sperm injection (ICSI) treatments. One wife developed ovarian hyperstimulation syndrome (OHSS), and 19 of 27 oocytes retrieved were subjected to ICSI but only one fertilized; the other wife had a normal response to ovarian stimulation, normal fertilization following ICSI, successful treatment and has recently delivered a live-born infant. The wife who suffered from OHSS has since had another ICSI cycle with a normal response to ovarian stimulation, a normal fertilization rate but no pregnancy. The only variable that determined the different rate of fertilization in the simultaneous ICSI cycles appears to be oocyte quality. While the results of frozen embryo replacement cycles following the decision to freeze all embryos following OHSS is generally satisfactory, it is important to counsel couples about the possible detrimental effects of OHSS on oocyte quality. PMID:9806288

Akagbosu, F; Marcus, S; Abusheikha, N; Avery, S; Brinsden, P

1998-09-01

338

Cryopreservation of Mammalian Oocyte for Conservation of Animal Genetics  

PubMed Central

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.

Prentice, Jennifer R.; Anzar, Muhammad

2011-01-01

339

Oocyte differentiation is genetically dissociable from meiosis in mice  

PubMed Central

Oogenesis is the process by which ovarian germ cells undertake meiosis and differentiate to become eggs. In mice, Stra8 is required for the chromosomal events of meiosis to occur, but its role in differentiation remains unknown. Here we report Stra8-deficient ovarian germ cells that grow and differentiate into oocyte-like cells that synthesize zonae pellucidae, organize surrounding somatic cells into follicles, are ovulated in response to hormonal stimulation, undergo asymmetric cell division to produce a polar body, and cleave to form two-cell embryos upon fertilization. These events occur without premeiotic chromosomal replication, sister chromatid cohesion, synapsis, or recombination. Thus, oocyte growth and differentiation are genetically dissociable from the chromosomal events of meiosis. These findings open to study the independent contributions of meiosis and oocyte differentiation to the making of a functional egg.

Dokshin, Gregoriy A.; Baltus, Andrew E.; Eppig, John J.; Page, David C.

2013-01-01

340

Production of Sry knockout mouse using TALEN via oocyte injection  

PubMed Central

Recently developed transcription activator-like effector nuclease (TALEN) technology has enabled the creation of knockout mice, even for genes on the Y chromosome. In this study, we generated a knockout mouse for Sry, a sex-determining gene on the Y chromosome, using microinjection of TALEN RNA into pronuclear stage oocytes. As expected, the knockout mouse had female external and internal genitalia, a female level of blood testosterone and a female sexually dimorphic nucleus in the brain. The knockout mouse exhibited an estrous cycle and performed copulatory behavior as females, although it was infertile or had reduced fertility. A histological analysis showed that the ovary of the knockout mouse displayed a reduced number of oocytes and luteinized unruptured follicles, implying that a reduced number of ovulated oocytes is a possible reason for infertility and/or reduced fertility in the KO mouse.

Kato, Tomoko; Miyata, Kohei; Sonobe, Miku; Yamashita, Satoshi; Tamano, Moe; Miura, Kento; Kanai, Yoshiakira; Miyamoto, Shingo; Sakuma, Tetsushi; Yamamoto, Takashi; Inui, Masafumi; Kikusui, Takefumi; Asahara, Hiroshi; Takada, Shuji

2013-01-01

341

Live imaging of GFP-labeled proteins in Drosophila oocytes.  

PubMed

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

Pokrywka, Nancy Jo

2013-01-01

342

Oocyte Cryostorage to Preserve Fertility in Oncological Patients  

PubMed Central

Thanks to the progress in oncostatic treatments, young women affected by cancer have a fairly good chance of surviving the disease and leading a normal post-cancer life. Quite often, however, polychemiotherapy and/or radiotherapy can induce ovarian damage and significantly reduce the content of follicles and oocytes inside the ovary, thus predisposing the patient to menstrual disorders, infertility, and precocious menopause. Several techniques have been proposed to preserve fertility in these patients; among them oocyte collection and cryopreservation prior to the oncostatic treatment has been widely applied in the last decade. The proper indications, the permitting conditions, the available hormonal stimulation protocols, as well as the effectiveness and limits of this option will be discussed herein, with a comprehensive and up-to-date review of the two techniques commonly used to cryostore oocytes, the slow-freezing technique and the vitrification technique.

Revelli, Alberto; Molinari, Emanuela; Salvagno, Francesca; Delle Piane, Luisa; Dolfin, Elisabetta; Ochetti, Simona

2012-01-01

343

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

PubMed

Intracytoplasmic sperm injection (ICSI) in the human is a very effective procedure which allows the fertilization of the majority of oocytes even in cases of extreme oligoasthenoteratozoospermia. Round-headed acrosomeless human spermatozoa, however, form an exception to this rule, because in about half of the couples with globozoospermia all oocytes remain unfertilized after injection. The incapacity of the spermatozoon to activate the oocyte following injection of round-headed spermatozoa could be the underlying mechanism. To investigate this hypothesis, activation rates of mouse oocytes injected with spermatozoa from a patient with globozoospermia were compared with those obtained after injection with normal spermatozoa. Of mouse oocytes surviving the injection with donor spermatozoa, 95% underwent activation, compared to none of the 88 mouse oocytes surviving the injection with round-headed spermatozoa. After fixation, prematurely condensed sperm chromosomes were found in these oocytes. Parthenogenetic activation of mouse oocytes (8% ethanol at 40 min after injection) injected with round-headed spermatozoa led to the activation of 96% of oocytes. These oocytes developed normally to the first mitosis and were fixed for analysis of the sperm karyotypes. The incidence of chromosomal abnormalities of round-headed spermatozoa (6%) was similar to that in spermatozoa from a fertile donor (9%). These data provide further information on the basic defect in cases of globozoospermia and demonstrate that globozoospermia is not associated with sperm karyotype abnormalities. PMID:8943524

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

1996-10-01

344

Relationship between follicle size and ultrasound-guided transvaginal oocyte recovery  

Microsoft Academic Search

We evaluated the relationship between follicle size and oocyte recovery (OR) using ultrasound-guided follicle aspiration. Thirty Holstein cows were subjected to OR without gonadotrophic therapy. Oocytes were recovered two to four times from each cow in a total of 67 aspiration sessions. Ovarian follicles with diameters ?4mm and >4mm were aspirated in separated groups. Recovered oocytes from each group were

Marcelo Marcondes Seneda; Cesar Roberto Esper; Joaquim Mansano Garcia; Joo Ademir de Oliveira; Roberta Vantini

2001-01-01

345

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

Microsoft Academic Search

Chromosomal abnormalities and abnormal embryonic development have previously been observed after human in vitro fertilization (IVF). Chromosomal abnormalities may arise not only after fertilization but even earlier during meiotic maturation of human oocytes in culture. Since chromosomal analysis is simple in oocytes during meiotic maturation, the chromosomal status was analyzed in oocytes which remained unfertilized in a human in vitro

Horst Spielmann; Christiane Krger; Manfred Stauber; Richard Vogel

1985-01-01

346

Effect of Season and Exposure to Heat Stress on Oocyte Competence in Holstein Cows1  

Microsoft Academic Search

Two experiments were conducted to evaluate sea- sonal variation in oocyte competence in Holstein cows and to test whether oocyte quality in summer is af- fected by the magnitude of heat stress. In the first experiment, ovaries of Holstein cows were collected from a slaughterhouse and used to harvest oocytes over 1 yr (n = 18 replicates). After in vitro

Y. M. Al-Katanani; F. F. Paula-Lopes; P. J. Hansen

2002-01-01

347

Identification of perilipin-2 as a lipid droplet protein regulated in oocytes during maturation.  

PubMed

Lipid droplet proteins regulate the storage and utilisation of intracellular lipids. Evidence is emerging that oocyte lipid utilisation impacts embryo development, but lipid droplet proteins have not been studied in oocytes. The aim of the present study was to characterise the size and localisation of lipid droplets in mouse oocytes during the periovulatory period and to identify lipid droplet proteins as potential biomarkers of oocyte lipid content. Oocyte lipid droplets, visualised using a novel method of staining cumulus-oocyte complexes (COCs) with BODIPY 493/503, were small and diffuse in oocytes of preovulatory COCs, but larger and more centrally located after maturation in response to ovulatory human chorionic gonadotrophin (hCG) in vivo, or FSH + epidermal growth factor in vitro. Lipid droplet proteins perilipin, perilipin-2, cell death-inducing DNA fragmentation factor 45-like effector (CIDE)-A and CIDE-B were detected in the mouse ovary by immunohistochemistry, but only perilipin-2 was associated with lipid droplets in the oocyte. In COCs, perilipin-2 mRNA and protein increased in response to ovulatory hCG. IVM failed to induce perilipin-2 mRNA, yet oocyte lipid content was increased in this context, indicating that perilipin-2 is not necessarily reflective of relative oocyte lipid content. Thus, perilipin-2 is a lipid droplet protein in oocytes and its induction in the COC concurrent with dynamic reorganisation of lipid droplets suggests marked changes in lipid utilisation during oocyte maturation. PMID:20883652

Yang, Xing; Dunning, Kylie R; Wu, Linda L-Y; Hickey, Theresa E; Norman, Robert J; Russell, Darryl L; Liang, Xiaoyan; Robker, Rebecca L

2010-01-01

348

Effects of in vitro maturation of monkey oocytes on their developmental capacity  

Microsoft Academic Search

The study of in vitro maturation (IVM) of rhesus monkey oocytes has important implications for biomedical research and human infertility treatment. In vitro-matured rhesus monkey oocytes show much less developmental potential than IVM oocytes of other species. Since about 1980 when rhesus monkey IVM, in vitro fertilization (IVF) and in vitro embryo culture (IVC) systems were established, numerous efforts have

P. Zheng

2007-01-01

349

Developmental competence of oocytes recovered from postmortem ovaries of the endangered Indian blackbuck (Antilope cervicapra).  

PubMed

The ability to rescue gametes from endangered or wildlife species and to subsequently produce viable embryos holds tremendous potential as a means to increase the population size of endangered or wildlife species. The objective of this study was to assess the meiotic and developmental competence of oocytes recovered from postmortem ovaries of the Indian blackbuck. Oocytes collected from the ovaries of dead blackbucks were allowed to mature in vitro and then tested for developmental potential by activation with ionomycin followed by treatment with 6-dimethylaminopurine. The average number of oocytes recovered per ovary was 10.9, and recovery of the oocytes did not depend on the presence or absence of the corpus luteum, on the side, size and weight of the ovaries or on the type of oocytes recovered. The proportion of good quality oocytes showing cumulus expansion and extrusion of the first polar body were 79.3% and 46.1% when cultured with gonadotropins. In vitro maturation studies indicated that the proportion of oocytes that reached MII stage was significantly higher when good quality oocytes (68%) were used compared with fair quality oocytes (48%) when cultured in the presence of gonadotropins. Furthermore, fifty eight percent of the in vitro matured oocytes cleaved, and thirteen percent of the cleaved oocytes developed into blastocysts. These findings suggest that the oocytes recovered from postmortem ovaries of the blackbuck can be utilized for production of embryos. PMID:20710122

Sambasiva Rao, Brahmasani; Uma Mahesh, Yelisetti; Lakshmikantan, Uthanda Raman; Suman, Komjeti; Venu Charan, Katari; Shivaji, Sisinthy

2010-12-01

350

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

PubMed

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

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

2013-06-01

351

Pure preovulatory follicular fluid promotes in vitro maturation of in vivo aspirated equine oocytes  

Microsoft Academic Search

In the mare, rates of fertilization and development are low in oocytes matured in vitro, and a closer imitation of in vivo conditions during oocyte maturation might be beneficial. The aims of the present study were, therefore, to investigate whether (1) equine oocytes can be matured in vitro in pure equine preovulatory follicular fluid, (2) priming of the follicular fluid

I. B Bgh; J Bzard; G Duchamp; M Baltsen; N Grard; P Daels; T Greve

2002-01-01

352

Fatty acid profile of oocytes and blastocysts during in vitro bovine embryo production  

Microsoft Academic Search

Lipid content in oocytes and embryos plays important roles both in energy metabolism during oocyte maturation, fertilization and early development, and in their cryopreservation. However the composition and function of lipids during in vitro embryo production is not well known. Fatty acid (FA) profiles in immature (IM) and mature (MAT) oocytes (3 samples each; 100 to 142 per sample) and

M. Lap; C. C. Marques; M. C. Baptist; M. I. Vasques; A. E. M. Horta; P. V. Portugal; R. J. B. Bess; R. M. Pereira

353

Cholesterol Depletion Disorganizes Oocyte Membrane Rafts Altering Mouse Fertilization  

PubMed Central

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.

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

2013-01-01

354

Cholesterol depletion disorganizes oocyte membrane rafts altering mouse fertilization.  

PubMed

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

Buschiazzo, Jorgelina; Ialy-Radio, Come; Auer, Jana; Wolf, Jean-Philippe; Serres, Catherine; Lefvre, Brigitte; Ziyyat, Ahmed

2013-01-01

355

Reactive oxygen species in bovine oocyte maturation in vitro.  

PubMed

The role of reactive oxygen species (ROS) in the in vitro maturation (IVM) of oocytes remains controversial. The aim of the present study was to determine possible fluctuations in ROS production during bovine oocyte IVM in the presence of different modulators of ROS generation. Cumulus-oocyte complexes were cultured in medium 199 (control) in the absence or presence of 0.6 mM cysteine, 1 mM 1-choro-2,4-dinitro benzene (CDNB), 2 microM diphenyliodonium, 0.5 mM N-nitro-L-arginine methyl ester or 10 microM sodium nitroprusside (SNP) at 39 degrees C, in 5% CO2 in humidified air for 22 h. In addition, the respiratory chain effectors potassium cyanide (KCN; 1 mM) and carbonyl cyanide m-chlorophenylhydrazone (0.42 microM) were used. Meiotic maturation was determined by the presence of MII. ROS production was evaluated in denuded oocytes at different time points as the ratio of 2',7'-dichlorodihydrofluorescein diacetate (DCHF-DA) to fluorescein diacetate (FDA). ROS levels, expressed as DCHF-DA:FDA, fluctuated throughout the 22 h of maturation depending on the treatment applied. At 12 h incubation in the presence of KCN and SNP, ROS levels were increased, whereas ROS levels after 12 h in the presence of cysteine were reduced (P<0.05). Both CDNB and SNP impaired meiotic progression. The higher metabolic activity demand during bovine oocyte maturation coincides with a concomitant reduction in ROS generation. These results suggest that 12 h would be a critical point for bovine oocyte IVM because it is closely related to the production of ROS at this time. PMID:19383267

Morado, Sergio A; Cetica, Pablo D; Beconi, Martha T; Dalvit, Gabriel C

2009-01-01

356

Optimization of cryoprotectant loading into murine and human oocytes.  

PubMed

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 dimethyl sulfoxide (Me(2)SO) into mouse oocytes at 23C. 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 Me(2)SO exposure time, revealing that neither shrinkage nor Me(2)SO exposure single-handedly impairs the fertilization and development rates. Thus, damage during one-step Me(2)SO addition appears to result from interactions between the effects of Me(2)SO toxicity and osmotic stress. We also investigated Me(2)SO loading into mouse oocytes at 30C. 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 Me(2)SO exposure time, using hypotonic diluents for cryoprotectant solutions. With this technique, 1.5 M Me(2)SO 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

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

2014-02-01

357

Lunar regolith penetrators and cutters  

NASA Technical Reports Server (NTRS)

An apparatus was designed and built for conducting simulation experiments on cutting tool penetration in the centrifuge. This equipment is mounted on the laminar container which is used for the regolith densification study, so that the end product of the latter, i.e., a regolith bed with the proper density profile, can be used directly for the penetration tests. In this apparatus, an etching tool is suspended through a pulley system by the action of a double acting air cylinder. By adjusting the air pressure acting on each side of the cylinder, the net downward force acting on the tool can be controlled. The penetration of the tool is measured by an LVDT. This apparatus was proof-tested in the centrifuge and is ready for use in conjunction with the regolith densification experiments.

Barnes, Frank; Sture, Stein

1991-01-01

358

Lunar regolith penetrators and cutters  

NASA Astrophysics Data System (ADS)

An apparatus was designed and built for conducting simulation experiments on cutting tool penetration in the centrifuge. This equipment is mounted on the laminar container which is used for the regolith densification study, so that the end product of the latter, i.e., a regolith bed with the proper density profile, can be used directly for the penetration tests. In this apparatus, an etching tool is suspended through a pulley system by the action of a double acting air cylinder. By adjusting the air pressure acting on each side of the cylinder, the net downward force acting on the tool can be controlled. The penetration of the tool is measured by an LVDT. This apparatus was proof-tested in the centrifuge and is ready for use in conjunction with the regolith densification experiments.

Barnes, Frank; Sture, Stein

1991-11-01

359

A Rapid Western Blotting Protocol for the Xenopus Oocyte  

PubMed Central

Often experimentalists require a quantitative assessment of the levels of heterologously expressed proteins to best interpret changed Ca2+ signaling patterns. Here, we detail a rapid and convenient western blotting method for individual Xenopus oocytes. The method exploits recently introduced rapid blotting systems, commercially available from Invitrogen (iBlot) or Bio-Rad (Trans-Blot Turbo). The key advantage is speed: from live cell to transferred membrane in <1 h. Therefore, oocytes can be conveniently processed for western blotting to assess relative expression levels, even after a long day of Ca2+ imaging experiments.

Lin-Moshier, Yaping; Marchant, Jonathan S.

2014-01-01

360

Artificial intelligence techniques for embryo and oocyte classification.  

PubMed

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

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

2013-01-01

361

A rapid Western blotting protocol for the Xenopus oocyte.  

PubMed

Often experimentalists require a quantitative assessment of the levels of heterologously expressed proteins to best interpret changed Ca(2+) signaling patterns. Here, we detail a rapid and convenient western blotting method for individual Xenopus oocytes. The method exploits recently introduced rapid blotting systems, commercially available from Invitrogen (iBlot) or Bio-Rad (Trans-Blot Turbo). The key advantage is speed: from live cell to transferred membrane in <1 h. Therefore, oocytes can be conveniently processed for western blotting to assess relative expression levels, even after a long day of Ca(2+) imaging experiments. PMID:23457341

Lin-Moshier, Yaping; Marchant, Jonathan S

2013-03-01

362

Mars surface penetrator: System description  

NASA Technical Reports Server (NTRS)

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

Manning, L. A. (editor)

1977-01-01

363

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

PubMed

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

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

2012-04-01

364

Vitrification of oocytes from endangered Mexican gray wolves (Canis lupus baileyi).  

PubMed

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

Boutelle, S; Lenahan, K; Krisher, R; Bauman, K L; Asa, C S; Silber, S

2011-03-01

365

Mammalian oocytes are targets for prostaglandin E2 (PGE2) action  

PubMed Central

Background The ovulatory gonadotropin surge increases synthesis of prostaglandin E2 (PGE2) by the periovulatory follicle. PGE2 actions on granulosa cells are essential for successful ovulation. The aim of the present study is to determine if PGE2 also acts directly at the oocyte to regulate periovulatory events. Methods Oocytes were obtained from monkeys and mice after ovarian follicular stimulation and assessed for PGE2 receptor mRNA and proteins. Oocytes were cultured with vehicle or PGE2 and assessed for cAMP generation, resumption of meiosis, and in vitro fertilization. Results Germinal vesicle intact (GV) oocytes from both monkeys and mice expressed mRNA for the PGE2 receptors EP2, EP3, and EP4. EP2 and EP4 proteins were detected by confocal microscopy in oocytes of both species. Monkey and mouse oocytes responded to PGE2 as well as agonists selective for EP2 and EP4 receptors with elevated cAMP, consistent with previous identification of EP2 and EP4 as G?s/adenylyl cyclase coupled receptors. Incubation of mouse GV stage oocytes with PGE2 delayed oocyte nuclear maturation in vitro, but PGE2 treatment did not alter the percentage of mouse oocytes that fertilized successfully. PGE2 treatment also decreased the percentage of monkey oocytes that resumed meiosis in vitro. In contrast with mouse oocytes, the percentage of monkey oocytes which fertilized in vitro was lower after treatment with PGE2. Monkey oocytes with intact cumulus showed delayed nuclear maturation, but fertilization rate was not affected by PGE2 treatment. Conclusions Monkey and mouse oocytes express functional PGE2 receptors. PGE2 acts directly at mammalian oocytes to delay nuclear maturation. Surrounding cumulus cells modulate the effect of PGE2 to alter subsequent fertilization.

2010-01-01

366

Drug penetration in solid tumours  

Microsoft Academic Search

To be most effective anticancer drugs must penetrate tissue efficiently, reaching all the cancer cells that comprise the target population in a concentration sufficient to exert a therapeutic effect. Most research into the resistance of cancers to chemotherapy has concentrated on molecular mechanisms of resistance, whereas the role of limited drug distribution within tumours has been neglected. We summarize the

Andrew I. Minchinton; Ian F. Tannock

2006-01-01

367

Projectile Penetration Through Rheopectic Fuels.  

National Technical Information Service (NTIS)

A program was conducted to study the effects on the penetration of .50 caliber projectiles though JP-4 jet fuel into which a visco-electic additive had been mixed. The additives tested were FR-3 obtained from the Western Company and B-200 obtained from th...

J. F. Lundeberg R. J. Bristow

1968-01-01

368

Penetrating injuries of the neck.  

PubMed Central

A review of 271 patients with penetrating wounds of the neck is presented. A policy of selective conservative management appears totally justified in view of the low mortality and morbidity in this series. Particular attention has been paid to the presentation and surgical approach to the injured vertebral artery. Images Fig. 3

Demetriades, D.; Stewart, M.

1985-01-01

369

GROUND PENETRATING RADAR ON MARS  

Microsoft Academic Search

In the past decade and in the next decade, several ground penetrating radar systems have been or will be launched toward Mars. GPR systems have been built or proposed for study of Mars and the Martian satellite, Phobos, from orbit, balloons, and rovers. The scientific problems include those of finding evidence of past or present life on Mars, unraveling the

Gary R. Olhoeft

1998-01-01

370

Milder Etchant For Penetrant Inspection  

NASA Technical Reports Server (NTRS)

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.

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

1990-01-01

371

A follow-up study with oocyte donors exploring their experiences, knowledge, and attitudes about the use of their oocytes and the outcome of the donation  

Microsoft Academic Search

Objective: To learn what information oocyte donors were given and wanted to have about the use of their oocytes and the outcome of the donation.Design: In-depth interviews.Setting: Participants recruited through IVF clinics, matching agency, the Internet, word of mouth, and newspaper ads.Participant(s): Thirty-three former oocyte donors and six women preparing to donate.Intervention(s): None.Main Outcome Measure(s): None.Result(s): Thirty-three former donors completed

Andrea L Kalfoglou; Gail Geller

2000-01-01

372

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

NASA Technical Reports Server (NTRS)

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.

Musacchia, X. J.

1972-01-01

373

Penetration below a convective zone  

NASA Technical Reports Server (NTRS)

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.

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

1994-01-01

374

Autoradiography in fetal golden hamsters treated with tritiated diethylnitrosamine  

SciTech Connect

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.

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

1981-04-01

375

Identifying new human oocyte marker genes: a microarray approach  

Microsoft Academic Search

The efficacy of classical IVF techniques is still impaired by poor implantation and pregnancy rates after embryo transfer. This is mainly due to a lack of reliable criteria for the selection of embryos with sufficient development potential. Several studies have provided evidence that some gene expression levels could be used as objective markers of oocyte and embryo competence and capacity

Stphan Gasca; Franck Pellestor; Sad Assou; Vanessa Loup; Tal Anahory; Herv Dechaud; John De Vos; Samir Hamamah

2007-01-01

376

Oocyte biology and genetics revelations from polar bodies  

Microsoft Academic Search

The enormous volume of the fertilized egg is attributable to the suppression of cleavage during oocyte growth and the unequal cleavages during the first and second meiotic divisions. The two products of these divisions are the diminutive polar bodies (PB), which contain a redundant set of chromosomes\\/chromatids plus cytoplasmic organelles. The PB have strictly limited but differential life spans; while

SA Gitlin; WE Gibbons; RG Gosden

2003-01-01

377

FT-IR Microspectroscopy on molecular building of Zebrafish oocytes  

NASA Astrophysics Data System (ADS)

Zebrafish oocytes growth causes relevant modifications in protein components; in fact, the proteolytic cleavage of vitellogenin, a large sex-specific phospholipoglycoprotein synthesized in the female liver, leads to three main yolk protein components (phosphovitin and lipovitellins 1, 2), present as a complex in the oocyte. FT-IR Microspectroscopy could have the potentiality of monitoring these biochemical changes during the maturation process. Representative spectra for I-II, IIIA, IIIB and IV classes oocytes (Hierarchical Clustering Analysis and Principal Component Analysis) were investigated to find specific vibrational patterns corresponding to different maturation degrees. On going from I-II to IV class oocytes, relevant spectral differences were found with III class exhibiting an intermediate spectroscopic behaviour. In particular, the increase of the convoluted band at 2925 cm -1 (CH 2 and CH 3 stretching modes), as well as of intensity band ratios at 2926/2954 cm -1 ( ?asym CH 2/CH 3), 2854/2873 cm -1 ( ?sym CH 2/CH 3) and 1452/1392 cm -1 ( ?CH/?COO), suggest longer lipidic chains; broadening of Amide I and II bands and absorptions at 1737 ( ?C dbnd O, phospholipids) and 1157 cm -1 ( ?C sbnd O and ?C sbnd OH carbohydrates) in lipovitellin and vitellogenin spectra, are present in III and IV classes.

Carnevali, Oliana; Conti, Carla; Ferraris, Paolo; Garavaglia, Maria Grazia; Gioacchini, Giorgia; Giorgini, Elisabetta; Rubini, Corrado; Sabbatini, Simona; Tosi, Giorgio

2009-12-01

378

Effect of semen preparation on IVF of prepubertal goat oocytes  

Microsoft Academic Search

The aim of these experiments was to study the effects of different methods of washing and selection of spermatozoa on the IVF of IVM oocytes from prepubertal goats. Fresh ejaculates from 3 males of proven fertility were processed according to the following treatments: 1) centrifugation in TALP, 2) centrifugation in sucrose-based Ficoll medium, 3) centrifugation in Percoll gradients at 40

M. J Palomo; D Izquierdo; T Mogas; M. T Paramio

1999-01-01

379

Experience of freezing human oocytes using sodium-depleted media  

Microsoft Academic Search

Human embryo cryopreservation techniques enable the storage of surplus embryos created during assisted reproduction procedures; however, the existence of these same surplus embryos has sparked further debate. What can be their fate once they are no longer desired by their parents or if the parents are deceased? Thus, the level of interest in the cryopreservation of oocytes has increased, as

R. Azambuja; A. Petracco; L. Okada; J. Michelon; F. Badalotti; M. Badalotti

2011-01-01

380

Tuboovarian Abscess Caused by Atopobium vaginae following Transvaginal Oocyte Recovery  

PubMed Central

A 39-year-old woman with tubarian sterility fell ill with acute pelvic inflammatory disease 2 months after transvaginal oocyte recovery. Laparotomy revealed a large tuboovarian abscess, from which Atopobium vaginae, an anaerobic gram-positive coccoid bacterium of hitherto unknown clinical significance, was isolated. The microbial etiology and the risk of pelvic infections following transvaginal punctures are discussed.

Geissdorfer, Walter; Bohmer, Christoph; Pelz, Klaus; Schoerner, Christoph; Frobenius, Wolfgang; Bogdan, Christian

2003-01-01

381

Protein Tyrosine Kinase Signaling During Oocyte Maturation and Fertilization  

PubMed Central

The oocyte is a highly specialized cell capable of accumulating and storing energy supplies as well as maternal transcripts and pre-positioned signal transduction components needed for zygotic development, undergoing meiosis under control of paracrine signals from the follicle, fusing with a single sperm during fertilization, and zygotic development. The oocyte accomplishes this diverse series of events by establishing an array of signal transduction pathway components that include a select collection of protein tyrosine kinases (PTKs) that are expressed at levels significantly higher than most other cell types. This array of PTKs includes cytosolic kinases such as SRC-family PTKs (FYN and YES), and FAK kinases, as well as FER. These kinases typically exhibit distinct patterns of localization and in some cases are translocated from one subcellular compartment to another during meiosis. Significant differences exist in the extent to which PTK-mediated pathways are used by oocytes from species that fertilize externally versus internally. The PTK activation profiles as well as calcium signaling pattern seems to correlate with the extent to which a rapid block to polyspermy is required by the biology of each species. Suppression of each of the SRC-family PTKs as well as FER kinase results in failure of meiotic maturation or zygote development, indicating that these PTKs are important for oocyte quality and developmental potential. Future studies will hopefully reveal the extent to which these factors impact clinical assisted reproductive techniques in domestic animals and humans.

McGinnis, Lynda K.; Carroll, David J.; Kinsey, William H.

2011-01-01

382

Protein tyrosine kinase signaling during oocyte maturation and fertilization.  

PubMed

The oocyte is a highly specialized cell capable of accumulating and storing energy supplies as well as maternal transcripts and pre-positioned signal transduction components needed for zygotic development, undergoing meiosis under control of paracrine signals from the follicle, fusing with a single sperm during fertilization, and zygotic development. The oocyte accomplishes this diverse series of events by establishing an array of signal transduction pathway components that include a select collection of protein tyrosine kinases (PTKs) that are expressed at levels significantly higher than most other cell types. This array of PTKs includes cytosolic kinases such as SRC-family PTKs (FYN and YES), and FAK kinases, as well as FER. These kinases typically exhibit distinct patterns of localization and in some cases are translocated from one subcellular compartment to another during meiosis. Significant differences exist in the extent to which PTK-mediated pathways are used by oocytes from species that fertilize externally versus internally. The PTK activation profiles as well as calcium signaling pattern seems to correlate with the extent to which a rapid block to polyspermy is required by the biology of each species. Suppression of each of the SRC-family PTKs as well as FER kinase results in failure of meiotic maturation or zygote development, indicating that these PTKs are important for oocyte quality and developmental potential. Future studies will hopefully reveal the extent to which these factors impact clinical assisted reproductive techniques in domestic animals and humans. PMID:21681843

McGinnis, Lynda K; Carroll, David J; Kinsey, William H

2011-01-01

383

Ultrastructural Changes in Bovine Oocytes Cryopreserved by Vitrification  

Microsoft Academic Search

Oocytes recovered from abattoir-derived ovaries were exposed to a cryoprotectant solution (DAP213: 2 M DMSO, 1 M acetamide, 3 M propanediol, and 10% fetal calf serum in tissue culture medium 199) for less than 20 s and vitrified either at the germinal vesicle (GV) stage or after maturation in vitro (IVM). Survival was assessed by fertilization and culture in vitro

E. Fuku; L. Xia; B. R. Downey

1995-01-01

384

Transcription Reactivation Steps Stimulated by Oocyte Maturation in C. elegans  

PubMed Central

Developing oocytes produce materials that will support early embryonic development, then cease transcription before fertilization. Later, a distinct transcription program is established in the embryo. Little is understood about how these global gene regulation transitions are effected. We have investigated in C. elegans how oocyte transcription is influenced by maturation, a process that releases meiotic arrest and prepares for fertilization. By monitoring transcription-associated phosphorylation of the RNA Polymerase II (Pol II) C-terminal domain (CTD), we find that oocyte transcription shuts down independently of maturation. Surprisingly, maturation signals then induce CTD phosphorylation that is associated specifically with transcription initiation steps, and accumulates to high levels when expression of the CTD phosphatase FCP-1 is inhibited. This CTD phosphorylation is also uncovered when a ubiquitylation pathway is blocked, or when maturation is stimulated precociously. CTD phosphorylation is similarly detected during embryonic mitosis, when transcription is also largely silenced. We conclude that oocyte maturation signals induce abortive transcription events in which FCP-1 may recycle phosphorylated Pol II, and that analogous processes may occur during mitosis. Our findings suggest that maturation signals may initiate preparations for embryonic transcription, possibly as part of a broader program that begins the transition from maternal to zygotic gene expression.

Walker, Amy K.; Boag, Peter R.; Blackwell, T. Keith

2007-01-01

385

Location of nuclear proteins on the chromosomes of newt oocytes  

Microsoft Academic Search

IN eukaryote cells chromosomal proteins are responsible for the organisational state of the DNA and the control of genetic expression, therefore knowledge of their location is an essential prerequisite for understanding chromosome function. Cytological localisation of chromosomal proteins is feasible using cells with giant chromosomes such as the oocytes of the newt Triturus cristatus carnifex where there are clearly defined

Sarah E. M. Scott; John Sommerville

1974-01-01

386

The portrayal of healthy women requesting oocyte cryo-preservation  

PubMed Central

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.

Mertes, H.

2013-01-01

387

Effects of lanosterol on in vitro maturation of porcine oocytes.  

PubMed

FF-MAS and T-MAS sterols, intermediaries in the cholesterol biosynthetic pathway present in all cells, may represent the physiological signal that instructs the oocyte to reinitiate meiosis. The purpose of this study was to examine the hypothesis that exogenous lanosterol could be included in the sterol biosynthetic pathway from acetate to cholesterol and induce resumption of meiosis in oocytes cultured in vitro. Porcine oocytes were in vitro matured in medium supplemented with different concentrations of lanosterol. First, after 22h of in vitro maturation, cumulus cells were recovery and Delta7-Reductase gene expression was quantified using real-time PCR. Second, after 44h of in vitro maturation, chromatin configuration was evaluated using Hoechst 33342. Lipid content was also evaluated at 22 and 44h of in vitro maturation using Nile red staining. The results showed that the addition of lanosterol increased the Delta7-Reductase gene expression and influenced resumption of meiosis using 50 and 100microM, as well as enhancing higher cytoplasmic lipid accumulation after in vitro maturation. The results demonstrate that exogenous lanosterol acts in the sterol biosynthetic pathway from acetate to cholesterol and it was responsible for a higher resumption of meiosis in porcine oocytes cultured in vitro. PMID:19473792

Marco-Jimnez, Francisco; Llobat, Lola; Vicente, Jos-Salvador

2010-02-01

388

Tuboovarian abscess caused by Atopobium vaginae following transvaginal oocyte recovery.  

PubMed

A 39-year-old woman with tubarian sterility fell ill with acute pelvic inflammatory disease 2 months after transvaginal oocyte recovery. Laparotomy revealed a large tuboovarian abscess, from which Atopobium vaginae, an anaerobic gram-positive coccoid bacterium of hitherto unknown clinical significance, was isolated. The microbial etiology and the risk of pelvic infections following transvaginal punctures are discussed. PMID:12791933

Geissdrfer, Walter; Bhmer, Christoph; Pelz, Klaus; Schoerner, Christoph; Frobenius, Wolfgang; Bogdan, Christian

2003-06-01

389

Tuboovarian Abscess Caused by Atopobium vaginae following Transvaginal Oocyte Recovery  

Microsoft Academic Search

A 39-year-old woman with tubarian sterility fell ill with acute pelvic inflammatory disease 2 months after transvaginal oocyte recovery. Laparotomy revealed a large tuboovarian abscess, from which Atopobium vaginae, an anaerobic gram-positive coccoid bacterium of hitherto unknown clinical significance, was isolated. The microbial etiology and the risk of pelvic infections following transvaginal punctures are discussed.

Christoph Bohmer; Klaus Pelz; Christoph Schoerner; Wolfgang Frobenius; Christian Bogdan

390

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

PubMed Central

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.

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

2012-01-01

391

The activity and copy number of mitochondrial DNA in ovine oocytes throughout oogenesis in vivo and during oocyte maturation in vitro  

PubMed Central

Mitochondria are responsible for the production of ATP, which drives cellular metabolic and biosynthetic processes. This is the first study to quantify the mtDNA copy number across all stages of oogenesis in a large monovulatory species, it includes assessment of the activity of mitochondria in germinal vesicle (GV) and metaphase II (MII) oocytes through JC1 staining. Primordial to early antral follicles (n = 249) were isolated from the sheep ovarian cortex following digestion at 37C for 1 h and all oocytes were disaggregated from their somatic cells. Germinal vesicle oocytes (n = 133) were aspirated from 3- to 5-mm diameter antral follicles, and mature MII oocytes (n = 71) were generated following in vitro maturation (IVM). The mtDNA copy number in each oocyte was quantified using real-time PCR and showed a progressive, but variable increase in the amount of mtDNA in oocytes from primordial follicles (605 205, n = 8) to mature MII oocytes (744 633 115 799, n = 13; P < 0.05). Mitochondrial activity (P > 0.05) was not altered during meiotic progression from GV to MII during IVM. The observed increase in the mtDNA copy number across oogenesis reflects the changing ATP demands needed to orchestrate cytoskeletal and cytoplasmic reorganization during oocyte growth and maturation and the need to fuel the resumption of meiosis in mature oocytes following the pre-ovulatory gonadotrophin surge.

Cotterill, Matthew; Harris, Sarah E.; Collado Fernandez, Esther; Lu, Jianping; Huntriss, John D.; Campbell, Bruce K.; Picton, Helen M.

2013-01-01

392

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.  

PubMed

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. PMID:2022950

Cecconi, S; Tatone, C; Buccione, R; Mangia, F; Colonna, R

1991-05-01

393

Improvement of embryonic development and production of offspring by transferring meiosis-II chromosomes of senescent mouse oocytes into cytoplasts of young mouse oocytes  

Microsoft Academic Search

PurposeThe effects of reciprocal transplantation of meiosis-II chromosomes between senescent and young mouse oocytes were evaluated\\u000a based on pre- and post-implantation development ability of resultant embryos.\\u000a \\u000a \\u000a \\u000a MethodsKaryoplasts including meiosis-II chromosomes of oocytes from senescent Rockefeller mouse\\/Ms-Rb(6, 15) females (10 to 12months,\\u000a age-related infertile mice) were transferred into cytoplasts of oocytes from young F1 females (3 to 5months). Reconstructed oocytes were

Akinori Mitsui; Midori Yoshizawa; Hiromichi Matsumoto; Emiko Fukui

2009-01-01

394

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

PubMed Central

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.

Davidson, Bryony; Murray, Alison A.

2013-01-01

395

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

PubMed Central

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.

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

2013-01-01

396

Thioredoxin-Interacting Protein Regulates Glucose Metabolism and Affects Cytoplasmic Streaming in Mouse Oocytes  

PubMed Central

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.

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

2013-01-01

397

CD81 and CD9 work independently as extracellular components upon fusion of sperm and oocyte  

PubMed Central

Summary When a sperm and oocyte unite into one cell upon fertilization, membranous fusion between the sperm and oocyte occurs. In mice, Izumo1 and a tetraspanin molecule CD9 are required for sperm-oocyte fusion as one of the oocyte factors, and another tetraspanin molecule CD81 is also thought to involve in this process. Since these two tetraspanins often form a complex upon cell-cell interaction, it is probable that such a complex is also formed in sperm-oocyte interaction; however, this possibility is still under debate among researchers. Here we assessed this problem using mouse oocytes. Immunocytochemical analysis demonstrated that both CD9 and CD81 were widely distributed outside the oocyte cell membrane, but these molecules were separate, forming bilayers, confirmed by immunobiochemical analysis. Electron-microscopic analysis revealed the presence of CD9- or CD81-incorporated extracellular structures in those bilayers. Finally, microinjection of in vitro-synthesized RNA showed that CD9 reversed a fusion defect in CD81-deficient oocytes in addition to CD9-deficient oocytes, but CD81 failed in both oocytes. These results suggest that both CD9 and CD81 independently work upon sperm-oocyte fusion as extracellular components.

Ohnami, Naoko; Nakamura, Akihiro; Miyado, Mami; Sato, Masahiro; Kawano, Natsuko; Yoshida, Keiichi; Harada, Yuichirou; Takezawa, Youki; Kanai, Seiya; Ono, Chihiro; Takahashi, Yuji; Kimura, Ken; Shida, Toshio; Miyado, Kenji; Umezawa, Akihiro

2012-01-01

398

Effects of Postmortem Interval on Mouse Ovary Oocyte Survival and Maturation  

PubMed Central

To study the time- and temperature-dependent survival of ovarian oocytes collected from postmortem carcass, ICR mice were killed and placed for different periods (0, 1, 2, 4, 6, 8 and 10 h) at different temperatures (25C, 4C and 37C). After preservation, oocyte morphology, germinal vesicle (GV) oocyte number, oocyte meiotic maturation percentage, mitochondrial distribution and intracellular glutathione (GSH) level were evaluated. The results showed no surviving oocytes could be collected by 2h, 6h, and 12 h after carcass preservation at 37C, 25C and 4C, respectively. The number of collected GV oocytes in the ovary deceased as the preservation time lasted at the same temperature. Meanwhile at the same point in time, the ratio of germinal vesicle breakdown (GVBD) and the first polar body emission (PBE) gradually reduced as preservation temperature increased. In addition, the percentage of abnormal mitochondrial distribution in the preserved oocytes was obviously higher than that in the control oocytes, while GSH level was not altered in collected oocytes. Unexpectedly, neither chromosome arrangement nor spindle organization was affected as long as the oocytes from preserved carcasses could complete maturation. These data are helpful for proper use of ovary oocytes from postmortem carcass of valuable individuals.

Zhang, Guang-Li; Ma, Jun-Yu; Sun, Quan; Hu, Meng-Wen; Yang, Xiu-yan; Gao, Si-Hua; Jiang, Guang-Jian

2014-01-01

399

Surgical removal of the pregastric pouch does not affect voluntary ethanol consumption in golden hamsters  

Microsoft Academic Search

Golden hamsters underwent either surgical removal of the pregastric pouch (PGX) or sham surgery. Following recovery from surgery, hamsters were given free access to 15% (v\\/v) ethanol solution for 40 days, and then to 30% ethanol solution for a further 32 days. PGX and sham-operated hamsters ingested similar amounts of absolute ethanol throughout the study, with intake exceeding 15 g\\/kg\\/day

David DiBattista

1995-01-01

400

Effects of wheel running on photoperiodic responses of Djungarian hamsters ( Phodopus sungorus )  

Microsoft Academic Search

Djungarian hamsters (Phodopus sungorus) were exposed to artificial short days either with access to a running wheel (RW) or without. Within 6weeks RW hamsters\\u000a considerably increased their body mass, whereas controls showed the typical body mass reduction. Estimation of paired testis\\u000a weights indicated a decelerated testis regression in RW hamsters. Subsequent locking of RWs for 9weeks led to a decline

Frank Scherbarth; Ines Petri; Stephan Steinlechner

2008-01-01

401

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

NASA Technical Reports Server (NTRS)

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.

Musacchia, X. J.

1973-01-01

402

Selective isolation and culture of a proliferating epithelial cell population from the hamster trachea  

Microsoft Academic Search

SummaryA reliable cell isolation technique was developed to allow the cultivation of cells from the hamster respiratory tract. Repeated\\u000a thermolysin treatments and gradient centrifugation yielded a cell culture completely free from contamination by fibroblasts.\\u000a Viable cells could be isolated from as little tissue as a single hamster trachea, but in vitro proliferation occurred only\\u000a if the hamster was less than

William E. Goldman; Joel B. Baseman

1980-01-01

403

Effect of mouse oocyte vitrification on mitochondrial membrane potential and distribution.  

PubMed

The effects of mouse oocyte vitrification on mitochondrial membrane potential and distribution were explored in this study. The collected mouse oocytes were randomly divided into vitrification and control groups. Ethylene glycol (EG) and dimethylsulphoxide (DMSO) were used as cryoprotectants in the vitrification group. The mitochondrial function and distribution in the oocytes were examined by using the fluorescent probes, JC-1 and Mito Tracker green. The results showed that the ratio of red to green fluorescence in mouse oocytes was significantly decreased after thawing in the vitrification group as compared with the control group (1.28 vs. 1.70, P<0.05). The percentage of polarized distribution of the mitochondria in oocytes was conspicuously reduced in the vitrification group when compared with the control group (31% vs. 63%, P<0.05). It was suggested that vitrification significantly affects the mitochondrial function and distribution in oocytes and reduces the potential of oocyte fertilization and embryo development. PMID:24496686

Lei, Tao; Guo, Na; Tan, Mei-hua; Li, Yu-feng

2014-02-01

404

Role of animal pole protuberance and microtubules during meiosis in sea cucumber Apostichopus japonicus oocytes  

NASA Astrophysics Data System (ADS)

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.

Pang, Zhenguo; Chang, Yaqing; Sun, Huiling; Yu, Jiaping

2010-05-01

405

Biological effect of focal alpha radiation on the hamster lung  

Microsoft Academic Search

Monodispersed 10-$mu$m diameter ZrO ceramic microspheres, ; containing varying amounts of PuO or PuO, were ; injected into the jugular vein of 100-day-old Syrian hamsters. These ; biologically inert microspheres lodged subsequently in pulmonary capillaries and ; remained static in position throughout the life span of the animals with no ; discernible inflammatory response. The numbers of microspheres injected ranged

D. M. Smith; E. C. Anderson; J. R. Prine; L. M. Holland; C. R. Richmond

1975-01-01

406

Effect of procarbazine on sperm morphology in syrian hamsters  

Microsoft Academic Search

The effect of procarbazine, an antineoplastic drug, on the reproductive system of male Syrian hamsters was studied. Exposure to procarbazine (5 daily doses ranging from 20 to 500 mg\\/kg body weight) resulted in 5? to 7.5?fold increase in sperm abnormalities, diminished sperm counts, and smaller testes within 4 wk. Transmission electron micrographs showed severe damage to the acrosomal plasma membrane

Harpal Singh; Thomas Kozel; Servon Jackson

1989-01-01

407

Genetics of Sex-linked yellow in the Syrian Hamster  

Microsoft Academic Search

Alternating patches of black and yellow pigment are a ubiquitous feature of mammalian color variation that contributes to camouflage, species recognition, and morphologic diversity. X-linked determinants of this patternrecognized by variegation in females but not in maleshave been described in the domestic cat as Orange, and in the Syrian hamster as Sex-linked yellow (Sly), but are curiously absent from other

Azita Alizadeh; Lewis Z. Hong; Christopher B. Kaelin; Terje Raudsepp; Hermogenes Manuel; Gregory S. Barsh

2009-01-01

408

Thymoquinone reduces hepatic glucose production in diabetic hamsters  

Microsoft Academic Search

The aim of this study was to elucidate the mechanisms underlying the glucose lowering effects of thymoquinone in streptozotocin (STZ)-induced diabetic hamsters in terms of hepatic glucose production. Diabetes was induced by intraperitoneal injection of 65mg\\/kg body weight of STZ. Treatment with thymoquinone commenced 4 weeks after induction of diabetes at a daily dose of 50mg\\/kg body weight by gastric

K. M. Fararh; Y. Shimizu; T. Shiina; H. Nikami; M. M. Ghanem; T. Takewaki

2005-01-01

409