bluuebunny/crossref_metadata_2025_split huggingface dataset:
Development and loss of the ability of mouse oolemma to fuse with spermatozoa
Authors: Zuccotti Maurizio , Piccinelli Anna , Marziliano Nicola , Mascheretti Silvia , Redi Carlo Alberto 
Published: September 2008

SummaryTo further our Knowledge on the mechanisms and molecules involved in mouse sperm–oocyte plasma membrane interaciton, exteraction, experiments were carried out to determine the stage during oogenesis at which an oocte acquires the capacity ot fuse with acrosome-reacted sperm. Zona-ferr oocytes 10 μm in diametes do not fuse with sperm. Oolemma fusibility is first acquired when the oocyte reaches about 20μm in diameter. Fusibility is maniatained even after fertilisation has accurred and is lost completely by the 4–cell stage.
F-actin is required for spindle anchoring and rotation inXenopusoocytes: a re-examination of the effects of cytochalasin B on oocyte maturation
Authors: Gard David Lynn , Cha Byeong-Jik , Roeder Amy Diane 
Published: September 2008

SummaryWe used confocal immunofluorescence microscopy to examine spindle migration, morphology and orientation during the maturation ofXenopusoocytes, in the presence or absence of cytochalasin B (CB), an inhibitor of actin assembly. Treatment with CB during maturation (10–50 μg/ml beginning 0–3h prior to addition of progesterone) disrupted the normal organisation of the novel MTOC and transient microtubule array (MTOC-TMA complex) that serves as the immediate precursor of the first meiotic spindle, suggesting that F-actin plays an important role in the assembly or maintenance of this complex. However, CB treatment did not block translocation of the MTOC-TMA complex to the oocyte cortex, suggesting that MTOC-TMA translocation is not dependent on an actin-based mechanism. Bipolar spindles were observed in CB-treated oocytes fixed during both M1 and M2. However, rotation of the M1 and M2 spindles into an orientation orthogonal to the oocyte surface was inhibited by CB. Rhodamine-phalloidin revealed a concentration of F-actin at the site of M1 spindle attachment, further suggesting that cortical actin is required for anchoring and rotation of the meiotic spindles. Finally, the incidence of M1 monasters was significantly increased in CB-treated oocytes, suggesting that interactions between the nascent M1 spindle and cortex are dependent on F-actin.
Early mouse preimplantation development is unaffected by microinjection of metallothionein antibodies
Authors: Ibánez Elena , Vidal Francesca , Hidalgo Juan 
Published: September 2008

SummaryPolyclonal antibodies that cross-react with rodent metallothionein I (MT I) and metallothionein II (MT II) were microinjected in 1-cell and 2-cell mouse embryos, into either the cytoplasm or the nucleus. Regardless of the experimental treatment, mouse embryo development in vitro was not affected and most of the embryos cleaved normally until the morula stage. The results suggest that metallothionein is not essential for normal mouse early preimplantational development, in agreement with recent studies in mice with inactivated MT I and MT II genes.
Beakers versus breakers: how fertilisation in the laboratory differs from fertilisation in nature
Authors: Mead Kristina S. , Epel David 
Published: September 2008

SummaryThe fertilisation of free-spawning invertebrates, mainly sea urchins, has been studied extensively during the last hundred years. However, results obtained fromin vitroexperiments do not always reflect what happens in the real world. Organisms in their natural habitats have a complex set of challenges, cues and behaviours to contend with during fertilisation and early development, factors that are normally not considered in the laboratory setting. This review examines recent work on fertilisation ecology and discusses the relevance of these results to the findings gleaned from laboratory research. Emphasis is placed on stresses associated with fertilisationin situ, and how responses to environmental stresses (such as from turbulence, oxidative stress, ultraviolet radiation and pathogens) might affect the fertilisation process.
Highly effective method of human oocyte activation
Authors: Levron Jacob , Cohen Jacques , Willadsen Steen 
Published: September 2008

SummaryFresh and aged unfertilised human oocytes were activated by electroporation and by exposure to isotonic solution of mannitol supplemented with low concentrations of calcium magnesium and chloride. Over 95% of the fresh oocytes were activated, all showing formation of one pronucleus and extrusion of the second polar body. Oocytes activated 1 and 2 days post-collection showed activation rates of 66.6% and 64.1%, respectively; however, the proportion of one-pronucleate oocytes in these groups was significantly lower (61.6% and 23.5%, respectively). There was no difference in the activation efficiency between the two activation modes. Twelve activated oocytes from the freshly collected group cleaved when left in culture. It is concluded that, in the human, a brief exposure to isotonic solution of mannitol with low concentrations of calcium, magnesium and chloride is a very effective activation stimulus.
Identification of porcine sperm plasma membrane proteins that may play a role in sperm–egg fusion
Authors: Ash Kendall , Berger Trish , Horner Catherine M. , Calvert C. Chris 
Published: September 2008

SummarySperm plasma membrane (PM) proteins that demonstrate affinity for egg PM preparations have the potential to be biologically important during sperm-egg binding and/or fusion. In this study four such proteins have been identified. To provide quantitative evidence for possible biological function, the large natural variation among different porcine sperm populations with regard to their ability to interact with the egg was compared with the relative binding of egg PM material to individual proteins. An aliquot from each of 24 porcine ejaculates was evaluated by the zona-free hamster ova bioassay and the remainder processed to yield sperm PM vesicles. Aliquots of sperm PM were solubilised, separated by SDS-PAGE, western blotted and probed with partially purified, biotinylated egg PM protein. Bound egg PM proteins were visualised on western blots by an avidin/biotin/horseradish peroxidase system and analysed by scanning laser densitometry. Four sperm PM proteins (62, 39, 27 and 7 kDa estimated molecular mass) were the predominant binders of egg PM. The amount of egg PM bound to the 62 kDa protein was significantly correlated with the ability of sperm from the 24 ejaculates to penetrate zona-free hamster ova (percentage of ova penetrated, p = 0.01, R = 0.65; number of penetrated sperm per ovum, p = 0.02, R = 0.63)
Ion channels from the mouse sperm plasma membrane in planar lipid bilayers
Authors: Labarca Pedro , Zapata Otilia , Beltrán Carmen , Darszon Alberto 
Published: September 2008

SummaryFusion of purified mouse sperm plasma membranes to planar lipid bilayers resulted in the insertion of three ion channel types. They could be discerned on the basis of their selectivity, conductance, gating and voltage-dependent properties. The presence of a previously reported large, Ca2+-selective channel was confirmed. Here, it is reported that the Ca21-selective channel from mouse sperm plasmamembrane displayed a pNa+/Pk+= 1.6 ± 0.2(n=4) and was blocked by micromolar concentrations of ruthenium red. Fusion yielded also a cation-selective channel (PNa+/Pk+= 2.5±0.3,n=3) with a main open conductance substate of 103 pS and a smaller open substate of 51 PS(600mM K+cis/100 mM Na+trans). The channel inserted into bilayers in two stable fashions: a high-activity mode (open probability = 0.57 ± 0.02, n=3), and a low activity mode (open probability <1%, n=4). In high mode, the channel displayed bursting kinetics and burst length was voltage independent. In addition, a perfectly anion-selective channel, with a slope conductance of 83 PS (600KCIcis/100KCItrans), was identified. It displayed a high, nearly constant open probability (∼0.90)in the 0 to –80 mV range.
Activation-dependent and activation-independent localisation of calmodulin to the mitotic apparatus during the first cell cycle of theLytechinus piçtusembryo.
Authors: Wilding Martin , Török Katalin , Whitaker Michael 
Published: September 2008

SummaryWe have used confocal microscopy and a fluorescent calmodulin probe to examine the mechanism of localisation of calmodulin during the first cell cycle of the sea urchin zygote. Using fluoresceincalmodulin, calmodulin can be observed within the nucleus and interphase astral microtubule arrays as cells approach mitosis. During mitosis, calmodulin redistributes to the mitotic apparatus and to condensed chromosomes. Quantitative analysis with reference to a control dye (fluorescein-dextran) shows that the distribution of calmodulin is specific. We used a competitive inhibitor of calcium-dependent calmodulin binding (Trp-peptide; Török & Trentham (1994)Biochemistry33, 12807–20) to test whether the cell cycle localisation of calmodulin was due to its binding to targets on activation. The Trp-peptide eliminates localisation of calmodulin within the nucleus. However, microtubule localisation persists in the presence of the Trp-peptide. These data show that calmodulin can localise by calcium (and hence activation)-dependent as well as calcium-independent mechanisms. This suggests that distinct mechanisms of localisation may be involved in the regulation of the differential functions of calmodulin, at least during the cell cycle.
Absence of an intracellular pH change following fertilisation of the mouse egg
Authors: Kline Douglas , Zagray James A. 
Published: September 2008

SummaryThe intracellular pH of the mouse egg was measured during fertilisation to determine whether an increase in pH accompanies activation of this mammalian egg. The pH-sensitive dye BCECF [2′,7′-bis-(2-carboxyethyl)-5(and-6)carboxyfluorescein] was introduced into the mouse egg by incubation in BCECF-AM or by microinjection of dextran-conjugated BCECF. The cells were also loaded with the DNA-specific fluorochrome Hoechst 33342 to confirm fertilisation by observation of Hoechst-stained, decondensing sperm heads in the cytoplasm. The ratio of emission intensities for the dye (494/440 nm excitation wavelengths) was monitored continuously with a photon-counting photomultiplier tube. There was no change in pH during or after fertilisation. Control eggs displayed the expected increase in pH when exposed to NH4C1. In other experiments, intracellular pH and intracellular Ca2+were monitored simultaneously during fertilisation. The eggs were injected with BCECF dextran and Fura dextran. Fluorescence emission was recorded at excitation wavelengths of 495 nm (BCECF, pH-sensitive wavelength) and 385 nm (Fura, Ca2+-sensitive wavelength). A decrease in emission intensity at 385 nm excitation clearly marked the repetitive Ca2+transients at egg activation. There was no change in the fluorescence emitted at 495 nm excitation, indicating an absence of any change in intracellular pH. These results indicate that intracellular alkalinisation of the cytoplasm does not accompany activation of this vertebrate egg.
The microenvironment created by non-blocking embryos in aggregates may rescue blocking embryos via cell–embryo adherent contacts
Authors: Sekirina Galina G. , Neganova Irina E. 
Published: September 2008

SummaryUnder our culture conditions, mouse embryos from the BALB/c inbred mouse strain develop successfully in culture only from the late 2-cell stage onwards (so-called 2-cell block), whether or not EDTA is added to the culture medium. (CBA × C57BL) F2embryos do not exhibit a 2-cell block. Medium conditioned by culture of non-blocking embryos from the 2-cell to the 8-cell stage did not improve the development of blocking embryos, nor did co-culture of blocking and non-blocking embryos, with or without conditioned medium. On the other hand phytohaemagglutinin (PHA)-assisted aggregation of an early 2-cell BALB/c embryo with five surrounding non-blocking F2embryos (2-cell or 8-cell) or five BALB/c 8-cell embryos allowed the early 2-cell BALB/c embryos to develop into blastocysts within 72 h. Aggregation of blocking BALB/c 2-cell embryos with each other had no ‘rescue’ effect. When blocking and non-blocking 2-cell embryos were aggregated together, an integrated blastocyst was formed; but when the early 2-cell BALB/c embryos were aggregated with non-blocking 8-cell embryos, the blocking embryos formed a separate small blastocyst, which nonetheless retained adherent contact with the non-blocking embryos throughout the culture period. Ultrastructural analysis showed that 2-cell embryos aggregated with the aid of PHA form close adherent cell contacts up to several micrometres in length.
Association between p34cdc2levels and meiotic arrest in pig oocytes during early growth
Authors: Hirao Yuji , Tsuji Youki , Miyano Takashi , Okano Akira , Miyake Masashi , Kato Seishiro , Moor Robert M. 
Published: September 2008

SummaryThe molecules involved in determining meiotic competence were determined in porcine oocytes isolated from preantral and antral follicles of different sizes. Oocytes isolated from preantral follicles had a mean diameter of 78 μm, contained diffuse filamentous chromatin in the germinal vesicle and were incapable of progressing from the G2to the M phase of the cycle even after 72 h in culture. Oocytes from early antral follicles had a mean diameter of 105 μm, showed a filamentous chromatin configuration and about half resumed meiosis but arrested at metaphase I (MI) when cultured. Oocytes from mid-antral (3–4 mm) and large antral follicles (5–6 mm) had mean oocyte diameters of 115 and 119 μm respectively, contained condensed chromatin around the nucleolus and progressed to metaphase II (MII) in 48% and 93% of instances respectively. Analysis of p34cdc2, the catalytic subunit of maturation promoting factor (MPF), by immunoblotting indicates that the inability of small (78 μm) oocytes to resume meiosis is due, at least in part, to inadequate levels of the catalytic subunit of MPF. On the other hand, the inability of intermediate-sized (105 μm) oocytes from antral follicles to complete the first meiotic division by progressing beyond MI appears not to be limited by levels of p34cdc2, which are maximal by this stage. We postulate that an inadequacy of molecules other than p34cdc2limits progression of MI to MII; the acquisition of these molecules during the final stages of growth may be correlated with the formation of the perinucleolar chromatin rim in the germinal vesicle.
Hypotaurine and taurine in gamete and embryo environments:de novosynthesis via the cysteine sulfinic acid pathway in oviduct cells
Authors: Guérin Pierre , Ménézo Yves 
Published: September 2008

SummaryTaurine and hypotaurine seem to be important compounds for sperm survival and capacitation, the fertilisation process and embryo development, and are present in both sperm and genital secretions. Hypotaurine has protective effects against peroxidative damage. We have established a simple method for hypotaurine quantification in sperm and genital secretions. The mean concentration of hypotaurine is significantly higher in bovine than in human spermatozoa and in seminal plasma. We observed that both molecules are secreted by cow, sow, goat and rabbit oviduct epithelial cell monolayers. In rabbit the release is ascorbic acid dependent. Goat oviduct epithelial cells are able to use the transsulfuration pathway to form hypotaurine and taurine from methionine. We were able to identify cysteine sulfinate decarboxylase (EC 4.1.1.29) activity in cow and goat tubal monolayers. Our results demonstrate that hypotaurine and taurine are secreted by oviduct epithelium, and synthesised by tubal cells via the cysteine sulfinic acid pathway. The data obtained emphasise the importance of hypotaurine and taurine for gamete maturation, fertilisation and early embryonic development.
Synergetic effects of epidermal growth factor and gonadotropins on the cytoplasmic maturation of pig oocytes in a serum–free medium
Authors: Wanga Weihua , Niwa Koji 
Published: September 2008

SummaryCumulus-enclosed or cumulus-free pig oocytes were cultured for 48 h in a serum-free maturation medium, TCM-199B containing 1 mg/ml polyvinylalcohol, supplemented with gonadotropins (PMSG + hCG) and/or epidermal growth factor (EGF), and inseminated with frozen-thawed ejaculated spermatozoa. Fourteen hours after insemination, the ability of oocytes to form male pronucleus (MPN) was examined. EGF significantly increased the ability of cumulus-enclosed oocytes to form MPN irrespective of the concentrations (1–100 ng/ml) tested. Gonadotropins had a greater effect than EGF on MPN formation in cumulus-enclosed oocytes, and a synergetic effect was observed when EGF and gonadotropins were added together. However, neither EGF nor gonadotropins exhibited a stimulatory effect on MPN formation in cumulus-free oocytes. These results indicate that the role(s) of both EGF and gonadotropins in stimulating cytoplasmic maturation of pig oocytes is mediated by cumulus.
The effect of temperature fluctuations on the cytoskeletal organisation and chromosomal constitution of the human oocyte
Authors: Almeida Paula A. , Bolton Virginia N. 
Published: September 2008

SummaryThe effect of temperature fluctuation on spindle integrity and chromosomal organisation in the human oocyte, and the consequences of such effects on the chromosomal constitution of resulting parthenotes, were investigated. A total of 340 oocytes were stained immunocytochemically with an antibody to α-tubulin, and 502 were activated parthenogenetically. Exposure of oocytes to room temperature for 2, 10 or 30 min caused disruption of the spindle in 77%(n = 26),72%(n = 18) and 89% (n = 19) of cases respectively, with evidence of chromosomal dispersal in 50%,56% and 52.6% respectively. These effects were reversed when oocytes were returned to 37°C after exposure to room temperature for 2 min, but not after 10 min or 30 min: Temperature reduction affected rates of parthenogenetic activation of oocytes (2 min: 67%,n= 27; 10 min: 68%,n= 28; 30 min; 54%,n= 35) and cleavege of resulting parthenotes, but only if oocytes were exposed to room temperature for 30 min (30 min: 53%,n= 19).There is a direct association between temperature-induced spindle damage in the oocyte(70%, 50 of 63) and chromosomal abnormalities in parthenotes developed from oocytes exposed to room temperature (56%,23 of 41;P< 0.01).
Calcium-dependent development of secondary cytostatic factor (2°CSF) fromXenopus laevisoocytes and zygotes
Authors: Zhang Jianshe , Masui Yoshio 
Published: September 2008

SummaryFresh cytosols extracted from unfertilised eggs ofXenopus laeviscontain a cytostatic factor (CSF) which arrests the cell cycle at metaphase when microinjected into cleaving blastomeres. This CSF is sensitive to Ca2+and is designated primary CSF (1°CSF). During storage of Ca2+-containing cytosols at 2°C, a stable CSF activity appears which is designated secondary CSF (2°CSF). In the present study, we report that 2°CSF activity can be induced in cytosols extracted from stage VI oocytes, unfertilised eggs, electrically activated eggs or blastulae, in the presence of Ca2+. Both the intensity and the rate of 2°CSF development are dependent on the concentration of Ca2+ions added to the cytosol. At Ca2+concentrations of 5–10 mM, 2°CSF activity reaches a maximum in about 7 days. Secondary CSF is relatively resistant to heat but loses all activity after 5 min at 70°C. When stored at −80°C, 2°CSF activity remains detectable for about 6 weeks. Cytological observations show that blastomeres arrested by microinjection of 2°CSF developed in cytosols of unfertilised eggs, activated eggs or blastulae contain metaphase chromosomes embedded in a bipolar spindle that has no asters developed at its poles. In contrast, blastomeres arrested by 2°CSF in cytosols of stage VI oocytes contain condensed chromosomes but no spindle is formed. The mechanisms of the development of 2°CSF in Ca2+-containing cytosols and its mode of action are discussed.
Characterisation and role of integrins during gametic interaction and egg activation
Authors: Nadai Céline de , Fenichel Patrick , Donzeau Michèle , Epel David , Ciapa Brigitte 
Published: September 2008

SummaryIt has recently been proposed that some of the processes induced by fertilisation in mammals may be mediated by integrins. By peforming immunofluorescence labelling and Western blots with antibodies directed against some of the α and β subunits of integrins, we show here the presence of some of these proteins in human and hamster oocytes. Among them, α2 and α5 were also present onin vitropreparations of sea urchin egg cortices. In addition, antibodies raised against these two proteins were the most effective at inhibiting attachment and fusion of human spermatozoa with hamster oocytes. We suggest that α2 and α5 integrin chains may be common mediators in adhesion-fusion mechanisms triggered by fertilisation. Using similar techniques, we show that eggs are rich three cytoskeletal proteins known to be linked to the β chain of integrins: talin, vinculin and α-actinin. Moreover, we found that talin and α-actinin were associated with proteins phosphorylated on tyrosine after fertilisation in sea urchin eggs. We suggest that integrins might be involved during fertilisation and trigger egg activation through cytoskeletal structures.
The influence of ploidy on the distribution of cells in chimaeric mouse blastocysts
Authors: Everett Clare A. , West John D. 
Published: September 2008

SummaryPrevious studies of tetraploid↔diploid mouse chimaeras and mosaics have revealed that tetraploid cells do not contribute equally to all tissues of the conceptus. In this study we have shown that, within 30 h of aggregating cleavage stage embryos, tetraploid cells were non-randomly distributed among different tissues of the early blastocyst. They were preferentially allocated to the mural trophectoderm regardless of cell size at the time of aggregation. This early effect may underlie the restricted distribution of tetraploid cells at later stages. We have demonstrated for the first time that ploidy can influence the relative position of blastomeres in the preimplantation embryo.
Comparative glucose and fructose incorporation and conversion by in vitro Produced bovine embryos
Authors: Guyader-Joly Catherine , Khatchadourian Chaqué , Ménézo Yves 
Published: September 2008

SummaryWe have investigated the quality of bovine IVM/IVF embryos co-cultured on Vero cells. Blastocyst cell numbers are very similar to those obtained in vivo, and higher than those obtained by co-culture with oviduct cells. The metabolism and conversion of fructose and glucose are not equivalent even though carbon dioxide production is similar and increasing from morula to blastocyst. Formation of free amino acids and incorporation into proteins are higher and faster for glucose than for fructose, but this conversion is rather stable with embryonic growth. Moreover, the by-products formed are not the same. Glucose at physiological concentrations (i.e.2 mM)seems to be a more appropriate fuel for the burst of embryonic development at the blastocyst stage in preparation for hatching.