Oocytes

The onset of downstream migration of European eels is accompanied by a cessation of feeding and the start of sexual maturation which stresses the link between metabolism and sexual maturation, also suggesting an important role for exercise. Exercise has been tested with eels in swim tunnels and was found to stimulate the onset of sexual maturation. In this study, we have investigated the interplay between migration and maturation in the field during the downstream migration of female silver eels. Temporal changes in migratory status and sexual maturation among silver eels of the upstream Rhine River system over 3 months of the migration season (August, September and October) were determined in biometrical parameters, plasma 17b-estradiol and calcium levels, oocyte histology and gonadal fat levels. Furthermore, the ecological relevant parameters age as determined by otolithometry and health aspects indicated by haematocrit, haemoglobin and swim-bladder parasite load were measured. Silver eels were estimated to be 14 years old. A strong temporal progression in migratory stage was shown over the months of downstream migration. Catches probably represented a mix of reproductive migrants and feeding migrants of which the ratio increased over time. Furthermore, this study confirmed our hypothesis linking the migratory stage to early maturation as indicated by enlargement of the eyes, oocyte growth and fat deposition in the oocytes, exactly the same changes as found induced by exercise but not ruling out environmental influences. Migrants show extensive fat uptake by the oocytes, probably stimulated by the swimming exercise. In addition, at least 83% of the silver eels in this spawning run may have suffered from negative effects of swim-bladder parasites on their swimming performance.

Protein phosphatase activities were analyzed in vivo in Xenopus oocytes. The dephosphorylation of microinjected 8 casein was inhibited when the tumor promoter okadaic acid was microinjected into oocytes. Inhibition was dose dependent and reversible; 50 % of activity was recovered 15-30 minutes after microinjection.

Most studies on preconception diagnosis published so far have used polymerase chain reaction (PCR) analysis to identify single gene defects. Although fluorescent DNA probes have been used to obtain a partial cytogenetic diagnosis of aneuploidies in first polar bodies without defined chromosome structures, the analysis of structural chromosome anomalies in the interphase nucleus is not adequate. We describe a procedure to obtain first polar body chromosome complements from hamster and human oocytes. In 63.6% (105 of 165) of hamster first polar bodies the chromosome complement showed a defined chromosome morphology and in 94.1% (16 of 17) of human oocytes fixed after follicular puncture it was possible to obtain high quality, well spread chromosome complements. First polar body chromosomes are fuzzy and shorter than oocyte chromosomes, but fluorescent in-situ hybridization results obtained in human first polar bodies clearly show that it is possible to detect whole chromosomes, centromeres and unique sequences, including the terminal regions of small chromosomes. This suggests that in fresh oocytes, DNA loss resulting from apoptotic chromosome fragmentation has not yet occurred. Using the procedure described, first polar bodies could be used to analyse the meiotic segregation of maternal structural abnormalities and to detect numerical chromosome anomalies in humans.

Obtaining karyotypes from human spermatozoa after microinjection into Syrian golden hamster oocytes is difficult and the hitherto reported results are unsatisfactory. This may be related to the injection and culture technique or to the high susceptibility of the hamster oocytes to undergo parthenogenetic activation or both. Therefore, we investigated the hamster oocyte-human sperm microinjection model using the following two approaches: (i) application of contemporary techniques for injection (touching the sperm tail) and culture (hamster embryo culture medium, HECM-3, 10% CO 2 ) and (ii) omission of Ca 2⍣ from the injection medium. Thus, in the first series of experiments, 252 hamster oocytes were injected with human spermatozoa. Among the 219 (87%) oocytes that survived the injection procedure, the mean percentages of male pronucleus formation [two pronuclei (2PN), two polar bodies (PB)], mitotic metaphase entry and sperm chromosome spreads were 41.4, 27.8 and 18.2% respectively. Analysis of the oocytes which failed to develop the male pronucleus following injection revealed that most of them had developed only the hamster female PN while the sperm nuclei were either intact or swollen (partially decondensed), indicating that failure of oocyte activation was not the likely reason for the failure of male PN formation in these oocytes. In the next series of experiments, sibling oocytes were alternately injected with spermatozoa suspended either in the regular (1.9 mM Ca 2⍣ ) or Ca 2⍣ -free injection medium (experiment set 2, n ⍧ 278). A significant improvement was noted in the mean percentages of oocytes with 2PN, 2PB, metaphase entry and sperm chromosome spreads in the Ca 2⍣ -free group versus the regular group (2PN, 2PB: 51 versus 36.6%, metaphase entry: 36.3 versus 26.9% and sperm chromosome spreads: 28 versus 20.4%; all P < 0.04). Thus, parthenogenetic activation appears to be one of the contributing factors for the failure of male PN formation after heterospecific hamster ICSI. From these experiments it can be concluded that application of the advanced injection and culture techniques and omission of Ca 2⍣ from the injection medium are promising for the routine application of the hamster oocyte microinjection for karyotyping of human spermatozoa with poor fertilizing capacity.

To start determining the nature of meiotic incompetence in goat oocytes, we have examined the expression of one of the potential pre-MPF subunits: the cyclin B 1 . We have been isolating a small DNA probe encoding the goat cyclin B 1 box to analyze the expression of the cyclin B 1 gene in competent and incompetent goat oocytes. This probe was easily obtained by polymerase-chain-reaction (PCR) on reversetranscribed mRNA from granulosa cells, using cyclin B specific primers derived from a bovine cDNA. The transcript corresponding to cyclin B 1 in goat granulosa cells is 1.8 kb. In situ hybridization analysis indicated that competent and incompetent oocytes contained cyclin B 1 mRNA, but also that active cyclin B 1 mRNA synthesis occured at the end of the growth phase, e.g., when oocytes progressed in the acquisition of meoitic competence. Western blot analysis, performed with a monoclonal anticyclin B 1 antibody, revealed in competent and incompetent oocytes a polypeptide of 65kDa corresponding to the goat cyclin B 1 protein. This pattern of cyclin B 1 expression further suggested that meiotic incompetence in goat oocytes could not be primarily correlated with a lack of cyclin B 1 protein as potential pre-MPF subunit, but to a limiting amount of this protein. Mol. Reprod. Dev. 47:222-228, 1997. r 1997 Wiley-Liss, Inc.

Vitrification, a method of rapid cooling, is an alternate cryopreservation method of oocytes and embryos. The present study was aimed to examine the effect of polyvinylpyrrolidone (PVP) on vitrification of buffalo oocytes. Cumulus oocyte complexes (COCs) with fully grown oocytes (120-130 µm in diameter) were aspirated from slaughtered buffalo ovaries for vitrification. COCs were treated with equilibration solution at room temperature for 5 min and then transferred to a vitrification solution for 1 min. Then the COCs were submerged into liquid nitrogen (-196C) for a while using cryotops. The COCs were thawed, diluted, and washed in a washing solution for 5 min, respectively. Vitrified oocytes were incubated for in vitro maturation (IVM) at 38.5C under an atmosphere of 5% CO2 in the air for 24 hrs. Cumulus cells surrounding the oocytes were removed mechanically, oocytes were fixed in acetic acid and ethanol, and stained with aceto-orcein to examine the meiotic stages of oocytes. The numbers of morphologically normal oocytes after vitrification were higher in 5% PVP than 0 and 10% PVP groups. A proportion of oocytes treated with 5% PVP reached the metaphase II (MII) stage while none of the oocytes from 0% and 10% PVP groupsdeveloped beyond anaphase I and metaphase I (MI) stages, respectively. These results show that PVP can be used as a cryoprotectant for the vitrification of buffalo oocytes.

The present study was designed to examine the influence of oocyte quality, culture media and gonadotropins on cleavage rate and development of in vitro fertilized buffalo embryos. Three experiments were conducted. In experiment 1, oocytes were classified by number of cumulus cell layers and morphology of the ooplasm as good, fair or poor. Oocytes were cultured for IVM, IVF and IVC in CR1aa medium. In experiment 2, good quality oocytes were cultured for maturation in: (1) CR1aa; (2) CR2aa; (3) TCM-199; (4) MEM or (5) RPMI-1640, and then fertilized using frozen thawed buffalo spermatozoa in CR1aa. The oocytes were cultured in the same medium used for maturation after fertilization. In experiment 3, oocytes were classified into three groups: group (1) was without gonadotropin and serve as a control; group (2) in which IVM medium was supplemented with 10 g/ml FSH and group (3) in which IVM medium was supplemented with 10 IU ml −1 eCG.

Cofilin is a small protein that belongs to the family of actin-depolymerizing factors (ADF). The main cellular function of cofilin is to change cytoskeletal dynamics and thus to modulate cell motility and cytokinesis. We have recently demonstrated that the actin cytoskeleton is involved in the modulation of Ca 2+ signalling in starfish oocytes. To extend these observations, we have explored whether cofilin influences Ca 2+ signalling in the oocytes. Here we show that microinjection of the functionally active cofilin alters the Ca 2+ signalling mediated by the three major second messengers, InsP 3 , NAADP, and cADPr. Cofilin intensifies the Ca 2+ signals induced by InsP 3 and NAADP, and delays those induced by cADPr. Furthermore, the injection of cofilin increases the Ca 2+ signals during hormone-induced oocyte maturation and fertilization. The results suggest that the dynamic regulation of F-actin by its binding proteins may play an important role in the modulation of intracellular Ca 2+ signalling.

Cryopreservation of oocytes by vitrification is a promising new technique for assisted human reproduction. Any new technical development must be accompanied with data concerning obstetric and perinatal outcome. This study analysed the obstetric and perinatal outcomes in 165 pregnancies and 200 infants conceived following oocyte vitrification cycles in three assisted reproduction centres. The results indicate that the mean birth weight and the incidence of congenital anomalies are comparable to that of spontaneous conceptions in fertile women or infertile women undergoing in-vitro fertilization treatment. These preliminary findings may provide reassuring evidence that pregnancies and infants conceived following oocyte vitrification are not associated with increased risk of adverse obstetric and perinatal outcomes.

Early preantral mouse follicles with a diameter of 110-160 m were cultured in vitro for 10 or 12 days. Mature oocytes were retrieved following hCG, and fertilization was attempted either by in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI). Two-cell and blastocyst formation rates and blastocyst cell numbers were compared between 10-day and 12day in vitro-matured oocytes versus in vivo-matured oocytes. Uncleaved IVF oocytes were subjected to chromosome analysis. The 2-cell formation rate was significantly improved by ICSI compared with IVF both in 10-day (72.1% versus 56.1%; P ‫؍‬ 0.03) and 12-day cultures (74.1% versus 54.5%; P ‫؍‬ 0.028). Cytogenetic analysis of uncleaved MII oocytes following IVF showed that about 30% of MII oocytes showed no sign of sperm penetration. The blastocyst formation rate was significantly lower in 12-day versus 10-day cultures, whether fertilization was by IVF (40.7% versus 62.4%, P ‫؍‬ 0.016) or by ICSI (32.5% versus 57.1%, P ‫؍‬ 0.035). Blastocyst cell numbers from IVF and ICSI 10-day groups were similar and both significantly higher (P Ͻ 0.001) than from IVF 12-day cultures. All above expressed values were significantly higher for in vivo-matured oocytes. In conclusion, fertilization of oocytes from in vitro-matured mouse preantral follicles can be optimized with ICSI, giving significantly higher 2-cell formation rates than IVF. Blastocyst formation rate was not influenced by the technique of fertilization but rather by the extent of the in vitro culture period. Best results on preimplantation development of oocytes for in vitro-matured preantral follicles were obtained with ICSI on oocytes from 10day in vitro cultures. embryo, follicle, in vitro fertilization, oocyte development

To investigate whether very short exposure of mature oocytes to sperm in vitro may affect the fertilization rates, embryo cleavage rates, and embryo quality between sibling oocytes in the same patient. Sibling oocytes of the same patient from 23 oocyte collection cycles were randomly allocated to the study group, with a 1-hr or 3-hr sperm-oocyte incubation, or the control group with the standard overnight gamete co-incubation. The fertilization rates, cleavage rates, and subsequent embryo quality were evaluated. Our results showed no statistically significant differences in fertilization rates, embryo cleavage rates, and quality of the embryos between the study group and the control group. Since the present study showed that long exposure of the oocyte to sperm has no advantage over short exposure, we prefer shortening the oocyte-sperm incubation period for reducing the negative effect induced by nonphysiologically high concentrations of spermatozoa.

Horse is a seasonal breeder and information on oocyte quality outside the breeding season is very limited. Ovaries obtained at the slaughterhouse are a convenient but often limited source of oocytes in this species. As the low quantity of ovaries leads to an intensive use of all available material, it would be useful to know whether ovaries collected during the non-breeding season are suitable for in vitro maturation (IVM). In an attempt to characterize the effect of season on oocyte quality, we investigated the permeability of the gap junctions (GJ) present between cumulus cells and oocytes because of their important role in oocyte growth and maturation. We also compared the effect of supplementing the maturation medium with bovine serum albumin (BSA) or oestrus mare serum (EMS). A total of 645 oocytes isolated from 158 and 154 ovaries collected during the breeding and the non-breeding season, respectively, were used in this study. Oocytes were matured for 30 h in TCM 199 supplemented either with 10% EMS or with 4 mg/ml BSA. The presence of permeable GJs between cumulus cells and oocytes was investigated with the injection of a 3% solution of the fluorescent dye Lucifer yellow into the ooplasm. No differences in efficiency of oocyte retrieval or oocyte meiotic competence were detected between oocytes collected during the breeding and non-breeding season. The vast majority (90%) of the oocytes collected during the breeding season had fully functional communications with their surrounding cumulus cells but such communications were completely interrupted in 55.3% of the oocytes collected during the nonbreeding season. During the non-breeding season, the proportion of oocytes whose communications with cumulus cells were classified as closed or intermediate at the end of maturation was lower in the group matured with BSA than with EMS (71.4 vs 97.7, p < 0.05). The same trend, although not statistically significant, was observed during the breeding season also. The presence of BSA caused an incomplete cumulus expansion during both seasons. Our data indicate that oocytes collected during the non-breeding season do not show any meiotic deficiency but lack active communication with the surrounding cumulus cells at the time of their isolation from the ovary. No data are available at present for determining the consequences on the developmental competence even if data from other species suggest that this is likely.

Purpose Oocyte cryopreservation may avoid many complications of human embryo freezing and provide future fertility for women undergoing cancer therapy. The objective of this study was to explore the application of intraand extracellular sugars in combination with small amounts of dimethylsulfoxide (DMSO) to human oocyte cryopreservation as an alternative approach. Methods Discarded human oocytes that were obtained from IVF patients under informed consent and IRB approval, were cryopreserved by slow cooling to −196°C after being randomly distributed into three groups: (i) DMSO control without intra-and extracellular sugar; (ii) extracellular sugar (raffinose) + DMSO; and (iii) intra-and extracellular sugar (trehalose and raffinose, respectively) + DMSO. Subsequently, all cryopreserved oocytes were thawed rapidly, and their survival was assessed by morphological criteria after 24 h of culture. Results A total of 71 oocytes were evaluated in three groups with survival rates of 88.5% (23/26), 68.2% (15/22), and 52.2% (12/23) for intra-and extracellular sugar +DMSO, extracellular sugar+DMSO, and DMSO control groups, respectively. Conclusion These results support the use of intra-and extracellular sugars as an alternative approach for cryopreservation of human oocytes.

table of contents † Introduction † Methods Literature search Study selection † Results Literature search Cumulus -oocyte complex Zona pellucida Perivitelline space Morphology of first polar body Shape of the oocyte Appearance of the whole ooplasm Presence of vacuoles and/or cytoplasmic inclusions Central granulation or centrally located granular cytoplasm Presence and morphology of the meiotic spindle Viscosity of the cytoplasm and the resistance of the cell membrane at ICSI Cumulative effect of multiple features † Discussion † Conclusion

Introduction 3. The zona pellucida, its composition and role during gamete interaction and fertilization 4. Sperm binding proteins / receptors for the zona pellucida 4.1. Galactosyltransferase 4.2. D-mannosidase 4.3. Protein tyrosine kinase (ZRK) 4.4. Lectin 5. Sperm binding proteins / receptors for the zona pellucida with a role in the acrosome reaction 6. Interaction of spermatozoa with the zona pellucida and development of the acrosome reaction 6.1. Physiological inducers of the acrosome reaction of human sperm 7. Signal transduction mechanisms and the zona pellucida-induced acrosome reaction 7.1. G i proteins 7.2. Intracellular free calcium 7.3. Polyphosphoinositides 7.4. Adenylyl cyclase activity and cAMP 7.5. Membrane tyrosine kinases 8. Intracellular proteases and the zona pellucida-induced acrosome reaction 9. Cellular biology of human sperm-zona pellucida interaction 9.1. Modulators of sperm-zona pellucida binding

As the human ovarian follicle enlarges in the course of a regular cycle or following controlled ovarian stimulation, the changes in its structure reveal the oocyte environment composed of cumulus oophorus cells and the follicular fluid (FF).In contrast to the dynamic nature of cells, the fluid compartment appears as a reservoir rich in biomolecules. In some aspects, it is similar to the plasma, but it also exhibits differences that likely relate to its specific localization around the oocyte. The chemical composition indicates that the follicular fluid is able to detect and buffer excessive amounts of reactive oxygen species, employing a variety of antioxidants, some of them components of the intracellular milieu.An important part is played by albumin through specific cysteine residues. But the fluid contains other molecules whose cysteine residues may be involved in sensing and buffering the local oxidative conditions. How these molecules are recruited and regulated to intervene su...

In an attempt to understand more fully processes that control the selection or recruitment of follicles for ovulation, the superovulation paradigm in combination with the androgen, 5a-dihydrotestosterone, or the antiandrogens, cyproterone or cyproterone acetate, was used in the immature mouse to alter the developmental potential of follicles destined to ovulate or to become atretic. Quantitative analysis of the numbers of eggs ovulated after one or more rounds of stimulation by pregnant mare's serum gonadotrophin followed by human chorionic gonadotrophin (PMSG-hCG), revealed a dose-dependent ovulation response to exogenous androgen and antiandrogen. Low dosages of androgen improved the ovulation response significantly. Large dosages of cyproterone and cyproterone acetate (100 mgkg body weight) generally decreased the ovulation number in gonadotrophin-injected mice, suggesting a role for androgen in preovulatory events that occur within the ripened follicle after the ovulatory stimulus (hCG) has been received. Low dosages of cyproterone, particularly 25 mgkg, significantly enhanced the ovulatory response, a phenomenon not observed for cyproterone acetate at this dosage. Radioimmunoassays of serum LH suggested that the differ-The present address of Vassie C. Ware is Box G

The aim of this study was to evaluate the effect of progesterone supplementation and stage of oestrous cycle on in vitro maturation (IVM) of canine oocytes. Oocytes were cultured in medium supplemented with 0, 2000, 4000 or 8000 ng progesterone ml(-1) (Expt 1; n=274 oocytes) or 0, 20, 200 or 2000 ng progesterone ml(-1) (Expt 2; n=789 oocytes). In Expt 3, oocytes (n=1202) were cultured in a bi-phasic system of meiotic arrest followed by IVM, both in the presence of 0, 20, 200 or 2000 ng progesterone ml(-1). Rates of meiotic resumption for Expt 1 ranged from 40.0% to 58.5%; there were no significant differences among groups. In Expt 2, rate of meiotic resumption was significantly lower in the 2000 ng progesterone ml(-1) treatment (35.5%) compared with the 200 ng progesterone ml(-1) treatment (54.0%; P&lt;0.05). There were no significant differences in rates of maturation to metaphase II among treatments in Expt 1 (1.8-8.6%) or Expt 2 (8.4-14.7%); however, oocytes collected from ovarie...

some specific features: very long transcription units, deregulated termination, and transcription of non-coding satellite repeats. Here, based on the modern view on a role of RNA interference machinery in regulation of genome expression, we suggest a mechanism of initiation of satellite DNA transcription and offer a novel interpretation of the 'classical' hypothesis that sought to explain the significance of widespread transcription during oocyte growth.

Abstract: Xenopus oocytes expressing high voltage-gated calcium channels (Cav) were exposed to formalin (0.5%, v/v, 5 min.) and the oocyte death and Cav currents were studied for up to 10 days. Cav channels were expressed with α1β1b and α2δ sub-units and the currents (IBa) were studied by voltage clamp. None of the oocytes was dead during the exposure to formalin. Oocyte death was significant between day 1 and day 5 after the exposure to formalin and was uniform among the oocytes expressing various Cav channels. Peak IBa of all Cav and A1, the inactivating current component was decreased whereas the non-inactivated R current was not affected by 5 min. exposure to formalin. On day 1 after the exposure to formalin, Cav1.2c currents were increased, 2.1 and 2.2 currents were decreased and 2.3 currents were unaltered. On day 5, both peak IBa and A1 currents were increased. Cav1.2c, 2.2 and 2.3 currents were increased and Cav2.1 was unaltered on day 10 after the exposure to formalin. Protein kinase C (PKC) may be involved in formalin-induced increase in Cav currents due to the (i) requirement for Cavβ1b sub-units; (ii) decreased phorbol-12-myristate,13-acetate potentiation of Cav2.3 currents; (iii) absence of potentiation of Cav2.3 currents following down-regulation of PKC; and (iv) absence of potentiation of Cav2.2 or 2.3 currents with Ser→Ala mutation of Cavα12.2 or 2.3 sub-units. Increased Cav currents and PKC activation may coincide with changes observed in in vivo pain investigations, and oocytes incubated with formalin may serve as an in vitro model for some cellular mechanisms of pain.

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

The concentrations of nucleoside triphosphate, inorganic phosphate and the yolk proteins, phosvitin and lipovitellin, have been monitored in living embryos of Xenopus Iwvis by 31P nuclear magnetic resonance (NMR) spectroscopy. The nucleoside triphosphate levels remain relatively constant at about 3.5 -4.5 nmol/embryo at least until the 'spontaneous movement' stage of development. By the swimming tadpole stage an inorganic phosphate resonance representing about 30 nmol/embryo becomes evident in the NMR spectrum. Computer manipulation also shows such a resonance, although smaller, to be present at a somewhat earlier developmental stage; these findings are confirmed biochemically. The major contribution to the NMR spectrum of oocytes, unfcrtilized eggs and early embryos is the yolk phosphoprotein resonance. On isolation of the yolk from the embryos it is possible to quantify the contribution to the NMR spectrum from the lipid-phosphate and protein-phosphate moieties of the yolk proteins. During development, as the yolk is used up, it is found that the protein-phosphate resonance disappears at a greater rate than the lipid-phosphate peak. The total phosphorus content of the embryo ( x 200 nmol/embryo) is shown biochemically to remain constant during development ; however, the total amount of phosphorus Abbreviutions. NMR, nuclear magnetic resonance; NTP, nucleoside triphosphate; Me,PI, tetramethyl phosphonium iodide.

P2X receptors for ATP have a wide range of physiological roles and comprise a structurally distinct family of ligand-gated trimeric ion channels. The crystal structure of a P2X4 receptor, in combination with mutagenesis studies, has provided a model of the intersubunit ATP-binding sites and identified an extracellular lateral portal, adjacent to the membrane, that funnels ions to the channel pore. However, little is known about the extent of ATP-induced conformational changes in the extracellular domain of the receptor. To address this issue, we have used MTSEA-biotinylation (N-Biotinoylaminoethyl methanethiosulfonate) to show ATP-sensitive accessibility of cysteine mutants at the human P2X1 receptor. Mapping these data to a P2X1 receptor homology model identifies significant conformational rearrangement. Electron microscopy of purified P2X1 receptors showed marked changes in structure on ATP binding, and introducing disulphide bonds between adjacent subunits to restrict intersubunit movements inhibited channel function. These results are consistent with agonist-induced rotation of the propeller-head domain of the receptor, sliding of adjacent subunits leading to restricted access to the upper vestibule, movement in the ion conducting lateral portals, and gating of the channel pore.

1. It is known that the a 4 -subunit is likely to occur in the brain predominantly in a 4 b 3 d receptors at extrasynaptic sites. Recent studies have revealed that the a 1 -, a 4 -, c 2 -and d-subunits may colocalize extrasynaptically in dentate granule cells of the hippocampus. In the present study, we characterized a series of recombinant GABA A receptors containing human (H) and rat (R) a 1 ⁄ a 4 -, b 2 ⁄ b 3 -and c 2S ⁄ d-subunits in Xenopus oocytes using the two-electrode voltage-clamp technique.

The anxiolytic compound etifoxine (2-ethylamino-6-chloro-4-methyl-4-phenyl-4H-3,1-benzoxazine hydrochloride) potentiates GABA A receptor function in cultured neurons (Neuropharmacology 39 (2000) 1523). However, the molecular mechanisms underlying these effects are not known. In this study, we have determined the influence of GABA A receptor subunit composition on the effects of etifoxine, using recombinant murine GABA A receptors expressed in Xenopus oocytes. Basal chloride currents mediated by homomeric β receptors were reduced by micromolar concentrations of etifoxine, showing that β subunits possess a binding site for this modulator. In oocytes expressing α 1 β x GABA A receptors (x = 1, 2 or 3), etifoxine evoked a chloride current in the absence of GABA and enhanced GABA (EC10)-activated currents, in a dose-dependent manner. Potentiating effects were also observed with α 2 β x , β x γ 2s or α 1 β x γ 2s combinations. The extent of potentiation was clearly β-subunit-dependent, being more pronounced at receptors containing a β 2 or a β 3 subunit than at receptors incorporating a β 1 subunit. The mutation of Asn 289 in the channel domain of β 2 to a serine (the homologous residue in β 1) did not significantly depress the effects of etifoxine at α 1 β 2 receptors. This specific pattern of inhibition/potentiation was compared with that of other known modulators of GABA A receptor function like benzodiazepines, neurosteroids, barbiturates or loreclezole.

Animals use diverse strategies to specify tissue lineages during development. A common strategy is to partition maternally supplied and localized lineage determinants into progenitor cells. The mouse embryo appears to use a different, more regulative strategy to specify the first three lineages: the epiblast (EPI: future embryo), the trophectoderm (TE: future placenta), and the primitive endoderm (PE: future yolk sac). These lineages are specified during two successive differentiation steps leading to formation of the blastocyst. Here, we review classic and contemporary models of early lineage specification in the mouse, and describe recent efforts to understand the molecular regulation of these events. We describe evidence that trophectoderm differentiation bears resemblance to the process of epithelialization and describe the importance of apical/basal protein complexes in regulating this process. Next, we present a revised model of PE specification, and describe evidence that PE cells in the inner cell mass sort out to occupy their ultimate position on the surface of the EPI. Finally, we describe factors that reinforce these lineages and three distinct stem cell types that can be isolated from them. Together, these mechanisms guide the differentiation of the first lineages of the mouse and thereby set up tissues that will be important for the first steps of embryonic body patterning.

The aim of genotoxicity tests in germ cells is to assess the impact of exposure to environmental mutagens that may represent a risk for the fertility or for the offspring of exposed subject. The comet assay on mature mouse oocytes is a simple, reproductive and rapid test to study primary DNA damage in oocytes. This test is used to complete toxicology assays applied in first line to somatic cells, and could find many applications in reproductive toxicology to study impact of environmental factors on female germ cells. We describe a practical application of comet assay in reproductive biology to assess the genotoxicity of cryoprotectants used at high concentrations in oocyte vitrification protocols. This test allowed us to demonstrate that dimethylsulfoxide and ethylene glycol are non-genotoxic for the mouse oocytes and led us to hypothesize a genotoxic effect of 1,2-propanediol (PrOH) at high concentrations after having observed induction of significant DNA damage on CHO cell line an...

This article describes a new organelle found in the cytoplasm of the growth stage fish oocytes. In particular, we describe its organization at the morphological level and investigate its composition by different cytochemical and immunocytochemical approaches with both light and electron microscope. The conclusion is that the body is a peculiar protein scaffold functioning as a temporary trap for the storage of rRNA in the mid to late growth stage oocytes. Its presence would be related to the reorganization of the mass of amplified rDNA in micronucleoli and to the consequent temporary stop in the rRNA synthesis. Mol.

The vanilloid receptor subunit 1, or transient receptor potential vanilloid 1 (TRPV1), integrates physical and chemical stimuli in the peripheral nervous system, playing a key role in inflammatory pain. Identification of potent TRPV1 antagonists is thus an important goal of current neuropharmacology. Herein, we describe the solid-phase synthesis of a series of indole-based peptoids (N-alkylglycines) and the biological activity of the peptoids as novel TRPV1 antagonists. The potency and selectivity of the compounds were determined by electrophysiological recordings in Xenopus oocytes. The most potent and selective noncompetitive TRPV1 antagonist of the series, compound 7, represents an interesting pharmacophoric structure for analgesic lead optimization.

Scientists are able to advance the frontiers of human reproduction by employing a variety of molecular biological techniques to understand the biological processes intricately linked to oocyte and ovarian follicle development. Despite an abundance of knowledge concerning essential pathways which may have critical roles in oogenesis and folliculogenesis, the repertoire of medications to treat female fertility problems remains limited to a few classes of drugs involved in the induction or suppression of folliculogenesis and ovulation; many of which are not specific in their drug actions and can give rise to complications during clinical application. This paper aims to review biomolecules and pathways (e.g. PI3K, WNT, MAPK) pertinent to ovarian follicular development and active in human oocytes, including those involved in communication between somatic cells within the ovary (cumulus cells, granulosa cells and thecal cells) and the oocyte itself. These biomolecules which are involved in the nuclear and cytoplasmic maturation of oocytes and the control of ovarian folliculogenesis have potential as targets for improved ovarian stimulation, optimisation of oocyte maturation, and as biomarkers of oocyte viability assessment.

BACKGROUND: The aim of our study was to evaluate the possibility of embryonic or somatic cell haploidization after fusion with intact or enucleated immature oocytes which were subsequently cultured in vitro. Embryonic or somatic cell nuclei do not undergo premature chromosome condensation when fused to intact or enucleated immature oocytes whose maturation is prevented by dibutyryl cyclic AMP (dbcAMP). The presence of dbcAMP permits, however, the completion of DNA replication in somatic cell nuclei. METHODS AND RESULTS: The chromosomes condensed when the reconstructed cells were released from the dbcAMP block. When somatic or embryonic nuclei were introduced into intact immature meiotically competent oocytes and subsequently cultured their chromosomes assembled on a common spindle with meiotic chromosomes and proceeded through the meioticlike division, judged according to the presence of the first polar body extruded. When embryonic cell nuclei were introduced into cytoplasts obtained from immature meiotically competent oocytes, polar bodies were extruded in about 75% of reconstructed cells but the metaphase plates were abnormal in almost all cases. When somatic cell nuclei were inserted into the above cytoplasts, polar bodies were extruded only very exceptionally and in these cells chromosomes were arranged in abortive metaphase plates. CONCLUSIONS: Our results suggest that somatic cell nuclei are unable to proceed through the reduction division (haploidization) when introduced into an immature oocyte meiotic cytoplasm.

In a two-microelectrode voltage clamp with Xenopus laevis oocytes, a petroleum ether extract of Acorus calamus rhizomes enhanced the GABA-induced chloride current through GABA A receptors of the α 1 β 2 γ 2S subtype by 277% ± 9.7% (100 μg/mL). β-Asarone (1), (+)dioxosarcoguaiacol (2), (+)-shyobunone (3), and (+)-preisocalamenediol (4) were subsequently identified as main active principles through HPLC-based activity profiling and targeted isolation. The compounds induced maximum potentiation of the chloride current ranging from 588% ± 126% (EC 50 : 65.3 ± 21.6 μM) (2) to 1200% ± 163% (EC 50 : 171.5 ± 34.6 μM) (1), whereas (−)isoshyobunone (5) and (−)-acorenone (6) exhibited weak GABA A modulating properties (5: 164% ± 42.9%; EC 50 : 109.4 ± 46.6 μM and 6: 241% ± 23.1%; EC 50 : 34.0 ± 6.7 μM). The relative configuration of 2 was established as 4R*8S*10R* by NOESY experiments and conformational analysis.

To analyze whether a change in three-dimensional structure of the zona pellucida could indicate suboptimal gamete quality. Prospective study. Women&amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;s general hospital. A total of 72 patients who gave informed consent. The birefringence of all oocytes was prospectively analyzed with an automatic user-independent polarization microscopy imaging system. Birefringence of the inner zona layer, preimplantation development, implantation, and pregnancy. In approximately one third of all gametes (244/712), the system&amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;s automatic detection of the inner zona layer did not succeed. This phenomenon was a negative predictor of compaction (P&amp;amp;amp;amp;amp;amp;amp;amp;amp;lt;0.01), blastulation (P&amp;amp;amp;amp;amp;amp;amp;amp;amp;lt;0.001), and pregnancy (P&amp;amp;amp;amp;amp;amp;amp;amp;amp;lt;0.001). In cases of successful zona imaging, the score based on the birefringence of the inner zona layer was a strong predictor of blastocyst formation but not of embryo quality or pregnancy (P&amp;amp;amp;amp;amp;amp;amp;amp;amp;gt;0.05). Interestingly, antagonist protocol resulted in lower zona scores as compared with the long protocol (P&amp;amp;amp;amp;amp;amp;amp;amp;amp;lt;0.05). Combining the information from both undetected and detected oocytes, zona imaging was a helpful tool in oocyte selection. This knowledge might further help to reduce both the time in culture and the number of concepti considered for transfer.

Genetic modifications causing germ cell death during meiotic prophase in the mouse frequently have sexually dimorphic phenotypes where oocytes reach more advanced stages than spermatocytes. To determine to what extent these dimorphisms are due to differences in male versus female meiotic prophase development, we compared meiotic chromosome events in the two sexes in both wild-type and mutant mice. We report the abundance and time course of appearance of structural and recombinationrelated proteins of fetal oocyte nuclei. Oocytes at successive days post coitus show rapid, synchronous meiotic prophase development compared with the continuous spermatocyte development in adult testis. Consequently, a genetic defect requiring 2-3 days from the onset of prophase to reach arrest registers pachytene as the developmental endpoint in oocytes. Pachytene spermatocytes, on the other hand, which normally accumulate during days 4-10 after the onset of prophase, will be rare, giving the appearance of an earlier endpoint than in oocytes. We conclude that these different logistics create apparent sexually dimorphic endpoints. For more pronounced sexual dimorphisms, we examined meiotic prophase of mice with genetic modifications of meiotic chromosome core components that cause male but not female sterility. The correlations between male sterility and alterations in the organization of the sex chromosome cores and X-Y chromatin may indicate that impaired signals from the XY domain (XY chromosome cores, chromatin, dense body and sex body) may interfere with the progression of the spermatocyte through prophase. Oocytes, in the absence of the X-Y pair, do not suffer such defects.

Oocytes from abattoir-sourced ovine ovaries were aspirated from 2-to 4-mm follicles using 25, 50 or 100 mmHg pressure and an aspiration pump, or a needle (20-G) and syringe (2.5 ml) and subjected to in vitro maturation, fertilization and culture to determine the effect of aspiration pressure on the number and quality of oocytes recovered, and early embryonic development. Oocyte recovery rate was similar between groups (range: 57.1-73.1%; p > 0.05). The number and percentage of grade I and II oocytes recovered was reduced for 100 mm (24.5 ± 3.6 and 31.1 ± 6.1%) compared with 25 mm (51.4 ± 7.0 and 60.2 ± 7.8%) and 50 mm pressure (40.8 ± 5.6 and 50.3 ± 4.4%) and a syringe (40.3 ± 12.0 and 45.2 ± 2.1%; p < 0.05). Oocyte cleavage was similar for all groups at 24 (range: 30.9-49.6%) and 48 h post-insemination (49.7-65.5%), but blastocyst formation (% cleaved oocytes) was lower for oocytes aspirated with 25 mm (37.8%) than 50 (69.2%) or 100 mm (67.2%) pressure, and a syringe (72.0%; p < 0.05). Embryo production efficiency (% of oocytes cultured developing to the blastocyst stage) was higher for oocytes aspirated with 50 mm (45.4%) and 100 mm pressure (43.8%) and a syringe (45.0%) than 25 mm pressure (18.8%; p < 0.05). These results demonstrate that the aspiration of ovine oocytes with an aspiration pressure of 50 mm, or aspiration with a needle and syringe are equally efficacious for the in vitro production of embryos.

Objective: Telomeres are DNA repeats which cap and protect chromosome ends, facilitate homologue pairing and chiasmata formation during early meiosis, and shorten with cell division and exposure to reactive oxygen to mediate aging. Early germ cells contain telomerase, a reverse transcriptase which adds telomeres to 3-prime DNA ends, but telomerase activity declines in oocytes, fixing telomere length earlier during development. Experimentally induced telomere shortening in mice disrupts meiosis, impairs chiasmata formation, halts embryonic cell cycles, and promotes apoptosis in embryos, a phenotype which mimics reproductive senescence in women. Ethical constraints limit study of human embryos to nondestructive assays, such as morphologic evaluation under transmission optics, but cytoplasmic fragmentation is a reliable marker of apoptosis. Study design: Study design consisted of observational study of effect of telomere length in human eggs on cytoplasmic fragmentation, and on other morphologic features of preimplantation embryos. To test the hypothesis that telomere shortening triggers apoptosis in human embryos, we evaluated telomere length as a predictor of cytoplasmic fragmentation in embryos from women undergoing in vitro fertilization. Results: Telomere length negatively predicted fragmentation in day 3 preimplantation embryos, after controlling for patient age and basal follicle stimulating hormone level. Telomere length did not predict other features of preimplantation embryo morphology. Conclusion: The finding that telomere length in human eggs predicts cytoplasmic fragmentation in embryos provides evidence that telomere shortening induces apoptosis in human preimplantation embryos, consistent with a telomere theory of reproductive senescence in women.

In this study we compared the effects of preculture storage of ovaries, IVM medium, a reduced O 2 atmosphere and duration of culture on in vitro maturation (IVM) of domestic cat oocytes. One randomly selected ovary of each pair (69 pairs) was stored in PBS at 10 8C for 16±24 h before oocyte recovery. The second ovary from each pair was used as a nonstored control. In Experiment I, we investigated the effect of culture media (TCM 199 versus SOF) and a reduced O 2 atmosphere (a humidi®ed gas atmosphere of either 5% CO 2 in air or 5% CO 2 :5% O 2 :90% N 2 ) on IVM of both stored and nonstored oocytes. In the second experiment, we compared timing of nuclear maturation of both stored and nonstored oocytes cultured for 17±18, 20±21, 24±26, 28±30, 33±34 or 42±45 h before being evaluated for meiotic status. In both, Experiments I and II, the recovery rate, quality and competence for maturation of oocytes originating from stored ovaries did not differ (P > 0:05) compared with nonstored. In Experiment I, neither culture medium (37.5 versus 43.2% of Metaphase II, respectively in TCM 199 versus SOF) or gas atmosphere (40.0 versus 32.5% of Metaphase II, respectively in 5% CO 2 in air versus 5% CO 2 :5% O 2 :90% N 2 ) affected oocyte maturation. In Experiment II, the mean proportion of oocytes achieving Metaphase II within 17±18 h of culture was 36.1% and did not signi®cantly increase (P > 0:05) over time up to 28 h. The highest proportion of oocytes (67.3%) reached Metaphase II stage after 42±45 h of culture. Therefore, we conclude that two``waves'' of nuclear maturation of cat oocytes can be distinguished. The ®rst wave takes place within 26 h and it is likely that most oocytes of this wave mature by 17±18 h; the second wave occurs after 28±30 h of IVM. It can be assumed that this double wave may re¯ect the presence of two Theriogenology 59 (2003) 1567±1574 (L. Ka Îtska-Ksia Îz Çkiewicz). 0093-691X/02/$ ± see front matter # 2002 Published by Elsevier Science Inc. PII: S 0 0 9 3 -6 9 1 X ( 0 2 ) 0 1 2 0 0 -1 oocyte populations with two different degrees of``prematuration'' which require different lengths of IVM. #