bluuebunny/crossref_metadata_2025_split huggingface dataset:
Stages leading to and following fusion of sperm and egg plasma membranes
Authors: Longo Frank J. , Cook Susan , McCulloh David H. , Ivonnet Pedro I. , Chambers Edward L. 
Published: September 2008

SummaryThe site of gamete interaction of electrophysiologically recordedLytechinus variegatuseggs, fixed with osmium tetroxide (O5O4) and/or glutaraldehyde (GTA) at varying intervals after the onset of the increase in membrane conductance induced by an attached sperm, has been examined by high-voltage and conventional transmission electron microscopy. Although GTA and a GTA-O5O4mixture iduced different electrical responses, specimens prepared with the two fixatives were ultrastructurally similar. In specimens observed within 5 s of the change in conductance, the acrosomal process projected through the vitelline layer and abutted the egg plasma membrane. A conspicuous layer of bindin surrounded the acrosomal process and connected the sperm to the egg's vitelline layer. In a fortuitous specimen fixed within 4 s following the change in conductance, the area of contact between the gamete plasma membranes possessed a trilaminar structure that separated the egg's and sperm's cytoplasms. The morphology of this area of contact was consistent with previously proposed intermediates of membrane fusion. Five to six seconds after the change in conductance, the sperm was connected to the egg via a narrow cytoplasmic bridge that consisted of the former acrosomal process and a projection of the egg cortex. The region of the bridge midway between the fused gametes was encircled by dense material that marked the site of sperm-egg fusion. Gamete interactions in which the activation potential was recorded (unclamped egg) were comparable in time and ultrastructure to events taking place in voltage-clamped eggs except for one major difference. Intact cortical granules (one to three) were observed beneath the tip of the incorporating sperm in unclamped eggs fixed following the onset of the activation potential, whereas all cortical granules dehisced in clamped eggs.
Metaphase arrest in newly matured or microtubule-depleted mouse eggs after calcium stimulation
Authors: Moses Ruth M. , Masui Yoshio 
Published: September 2008

SummaryIn mouse eggs arrested at meiotic metaphase II, the increase in intracellular calcium that results from fertilisation induces nuclear formation in both newly ovulated and older eggs. In contrast, the calcium increase that results from exposure to the calcium ionophore A23187 induces nuclear formation in older, but not young, newly ovulated eggs. When treated with the microtubule inhibitor colcemid, and fertilised, young eggs remained at metaphase, but many older eggs formed nuclei, although older eggs treated with colcemid and A23187 remained at metaphase. However, young A23187-treated eggs, young colcemid-treated fertilised eggs, and older colcemid- and A23187-treated eggs, formed nuclei when treated, in addition, with the protein synthesis inhibitor cycloheximide, or the protein kinase inhibitor 6-dimethylaminopurine (6-DMAP). The possibility is discussed that metaphase in newly matured eggs and microtubule-depleted eggs may be maintained by similar mechanisms involving short-lived phosphorylated proteins.
Mouse sperm fertilising capacity following subzonal microinjection is dependent on sperm washing and response to solubilised zonae pellucidae
Authors: Kaplan Orly Lacham , Trounson Alan 
Published: September 2008

SummarySpermatozoa obtained from hybrid male mice were treated with solubilised zonae pellucidae after a period of capacitationin vitroto induce the acrosome reaction. Single spermatozoa were selected and microinjected into the perivitelline space of mature oocytes. A high proportion of the spermatozoa acrosome-reacted affter treatment with solubilised zonae (63%). However, the fertilisation rate (37%) after subzonal microinjection of oocytes by the treated spermatozoa was not different to the fertilisation rate (40%) of oocytes microinjected with untreated spermatozoa which had a lower rate of acrosome reaction (39%). When spermatozoa were washed by high-speed centrifugation before treatment with solubilised zonae pellucidae and subzonal microinjection, the fertilisation rate (68%) was significantly higher (p< 0.001) than that (29%) for occytes microinjected with untreated spermatozoa and was found to be correlated with a high acrosome reaction rate (74%) (r= 0.8). The washing of spermatozoa by centrifugation itself did not increase the acrosome reaction rate or fertilisation rate of oocytes after microinjection. The results of this study suggests that some modifications other than the acrosome reaction are needed to enable capacitated mouse spermatozoa to fuse with the oocyte plasma membrane. These modifications were achieved by washing spermatozoa by high-speed centrifugation and the replacement of the supernatant with fresh culture medium used for capacitation. Induction of the acrosome reaction by solubilised zonae pellucidae following this treatment leads to a high fertilisation rate of oocytes by subzonal sperm microinjection.
Characterisation of isolated egg cells,in vitrofusion products and zygotes ofZea maysL. using the technique of image analysis and confocal laser scanning microscopy
Authors: Tirlapur Uday K. , Kranz Erhard , Cresti Mauro 
Published: September 2008

SummaryChanges in membrane Ca2+, calcium receptor protein calmodulin, endoplasmic reticulum (ER), mitochondria and cellulose in unfixed, living, isolated egg cells and fusion products of pairs of one egg and one sperm cell ofZea maysL. have been investigated using chlorotetracycline, fluphenazine, immunocytochemical techniques, 3,3'dihexyloxa-carbocyanine iodide (DiOC6(3)) and calcofluor white in conjuction with computer-controlled video image analysis. In addition, confocal laser scannig microscopy has been used in conjuction with ethidium bromide to detect the nature and location of the sperm cell nuclear chromatin before and after karyogamy. Digitised video images of chlorotetra cycline (CTC) fluorescence reveal that egg cells contain high levels of membrane Ca2+in organelles present around the nucleus while the cytosolic signal is relatively low. Intense CTC fluorescence is invariably present just below the plasma membrane of egg cells and a certain degree of regionalised distribution of Ca2+in cytoplasm is also discrnible. Similarly, the fluphenazine (FPZ)-detectable calmodulin (CaM) and that localised immunocytochemically using monoclonal anti-CaM antibodies reveal high levels of Cam in the vicinity of the nucleus in egg cells. Only a few ER profiles and mitochondria could be visualised in the egg cell and no calcofluor fluorescence could be detected. Followingin vitrofertilisation of single isolated eggs substantial changes in the Ca2+levels occur which include an increase in the membrane Ca2+of the fusion product, particularly in the cytosol and around the nucleus. Unlike in the eggs the fine CTC fluorescence signal below the plasma membrane is not detectable in the fusion products. Compared with isolated egg cell protolasts an increase in the CaM level in the cytoplasm was observed in the fusion products. There is a slight increase in the CaM level in the cytoplasm was observed in the fusion products. There is a slight increase in the fluorescene around the fusion product is visible after 16 h in in culture. The sperm cell chromatin in the fusion product is highly condensed, unlike that of the egg cell, and confocally imaged serial optical sections of thein vitrofusion product reveal the occurrence of karyogamy 35 min following gamete fusion. First visual evidence for intermingling of sperm nuclear chromatin in the zygotic nuclei is also provided.
Molecular structure and function of CD4 on murine egg plasma membrane
Authors: Guo Mao Wu , Watanabe Toshiki , Mori Etsuko , Mori Tsuneatsu 
Published: September 2008

SummaryIn the present study, the expression of the CD4 molecule on murine egg plasma membrane was confirmed by the indirect immunofluorescence (IIF) method. The full-length CD4 cDNA from murine eggs was synthesised by the reverse transcriptase-polymerase chain reaction (RT-PCR) method and its authenticity verified by Southern blot hybridisation using an end-labelled internal oligonucleotide. The results of DNA sequencing showed that the nucleotide sequence of the cDNA of CD4 from murine egg mRNA was identical to that of immune T cells. To demonstrate the direct interaction of CD4 from murine egg with murine sperm cells bearing MHC (major histocompatibility complex) class II molecule, we employed a baculovirus expression system to generate CD4 on the surface ofSpodoptera frugiperda(Sf9) cells. Expression of CD4 on Sf9 cells infected with autographa californica nuclear polyhedrosis virus (AcNPV)-CD4 was demonstrated by IIF and immunoblotting. The CD4-expressing Sf9 cells adhered to MHC class II-bearing sperm cells since the adhension was specifically blocked by anti-CD4 monoclonal antibody (mAb) or anti-monomorphic region of MHC class II mAb. Taking our previous and present experimental results together, they strongly suggest that intercellular membrane adhesion between two gametes at the fusion step in fertilisation is mediated by the MHC class II molecule located on the posterior region of the sperm head and the CD4 molecule on egg plasma membrane.
Sulphydryl reagent iodoacetamide inhibits progression of meiosis and sperm transformation in mouse oocytes fertilisedin vitro
Authors: Maleszewski Marek 
Published: September 2008

SummaryThe effect of iodoacetamide (IA), a sulphydryl blocking agent, on fertilisation in the mouse was examined by transferring zona-free oocytes into IA (500 μM)-containing medium at various times after insemination. When inseminated oocytes were transferred into IA medium 10 min after insemination, the oocyte chromosomes remained aggregated in one or two masses and the sperm nucleus failed to decondense. When oocytes were transferred during the second meiosis, oocytes meiosis was more or less arrested. The sperm nucleus decondensed but remained blocked at an early stage of decondensation. These observations suggest that thiol groups in the oocyte's cytoplasm and perhaps microtubules of the meiotic spindle play crucial roles in the completion of meiosis and the transformation of sperm nucleus into pronucleus.
Cortical changes in starfish (Asterina pectinifera) oocytes during 1-methyladenine-induced maturation and fertilisation/activation
Authors: Longo Frank J. , Woerner Mark , Chiba Kazuyoshi , Hoshi Motonori 
Published: September 2008

SummaryMaturation of the starfish oocyte cortex to produce an effective cortical granule reaction and fertilisation envelope is believed to develop in three phases: (1) pre-methyladenine (1-MA) stimulation; (2) post-1-MA stimulation, pregerminal vesicle breakdown; and (3) post-germinal vesicle breakdown. The present study was initiated to identify what each of these phases may encompass, specifically with respect to structures associated with the oocyte cortex, including cortical granules, microvilli and vitelline layer. 1-MA treatment brought about an orientation of cortical granules such that they became positioned perpendicular to the oocyte surface, and an ∼ 4-fold decrease in microvillar length. A-23187 activation of immature oocytes treated with (10 min; pregerminal vesicle breakdown) or without 1-MA resulted in a reduction in cortical granule number of 21% and 41%, respectively (mature oocytes underwent a 96% reduction in cortical granules). Elevation of the fertilisation envelope in both cases was significantly retarded compared with activated mature oocytes. In activated mature oocytes, the vitelline layer elevated 20.0 ± 5.4 μm from the egg's surface, whereas in immature oocytes treated with just A-23187 or with 1-MA (10 min) and A-23187, it lifted 0.35 ± 0.1 and 0.17 ± 0.04 μm, respectively. The fertilisation envelopes of activated (or fertilised) immature oocytes also differed morphologically from those of mature oocytes. In activated, immature oocytes, the fertilisation envelope was not uniform in its thickness and possessed thick and thin regions as well as fenestrations. Additionally, it lacked a complete electron-dense stratum that characterised the fertilisation envelopes of mature oocytes. The nascent perivitelline space of immature oocytes was also distinguished by the presence of numerous vesicles which appeared to be derived from microvilli. Differences in the morphology of cortices from activated (fertilised) and non-activated, immature and mature oocytes substantiate previous investigations demonstrating three phases of cortical maturation, and are consistent with physiological changes that occur during oocyte maturation, involving ionic conductance of the plasma membrane, establishment of slow and fast blocks to polyspermy and elevation of a fertilisation envelope.
Mast cell-Leydig cell relationships in the testis of the lizard podarcis s. sicula Raf: thermal manipulation, ethane 1,2-dimethane sulphonate (EDS) and sex hormone treatment
Authors: Minucci Sergio , Vitiello Italia Izzo , Marmorino Concetta , Di Matteo Loredana , Baccari Gabriella Chieffi 
Published: September 2008

SummaryMast cells of connective tissue type are scattered in the interstitial compartment of the lizard Podarcis s. sicula. Their number varies during the year, showing peaks in spring and in winter, respectively.Thermal manipulation affects mast cell number (MCN): high temperature decreased MCN in both January and May, while low temperature increased MCN only in January. Ethane dimethane sulphonate, a toxin which specifically destroys Leydig cells, induced an increase in MCN on days 3 and 7 of treatment. Oestradiol treatment provoked a strong increase in MCN that was blocked by tamoxifen. Blocking androgen receptors with cyproterone acetate resulted in an increase in MCN, while testosterone injection provoked a strong decrease. These results suggest a relationship between the presence of mature Leydig cells and mast cell proliferation and/or differentiation.
The role of microtubules and inositol triphosphate induced Ca2+release in the tyrosine phosphorylation of mitogen-activated protein kinase in extracts ofXenopus laevisoocytes
Authors: Duesbery N.S. , Masui Y. 
Published: September 2008

SummaryMicrosomal fractions ofXenopusoocytes release preloaded45Ca2+when treated with inositol triphosphate (InsP3). The effective concentration of InsP3required for half-maximal release (EC50) is 59 nM and maximal release occurs at ∼ 2 μM InsP3. Uptake and release of45Ca2+are not altered by the catalytic subunit of protein kinase A, dibutyrl cyclic adenosine monophosphate, protein kinase A peptide inhibitor or nocodazole. In contrast, taxol decreases the sensitivity of the microsomal fraction to InsP3, shifting the EC50for InsP3-induced Ca2+release from 59 to 259 nM. In lysates of oocytes, InsP3-induced Ca2+release causes the tyrosine phorphorylation of a 42000 (Mr42k) protein identified as 42k mitogen-activated protein (MAP) kinase. InsP3-induced tyrosine phosphorylation of MAP kinase is prevented by BAPTA and taxol, but not by nocodazole. Thus, microtubule polymerisation modifies InsP3-induced Ca2+release, thereby inhibiting phosphorylation of MAP kinase.
First meiosis of early dictyate nuclei from primordial oocytes in mature and activated mouse oocytes
Authors: Czolowska Renata , Tarkowski Andrzej K. 
Published: September 2008

SummaryNuclei of diplotene (dictyate) primordial oocytes (PO) were transferred to metaphase II oocytes and to activated mouse oocytes using cell fusion techniques. In a metaphase II oocyte, the PO nucleus condenses within 2–3 h to bivalents which become arranged on the first meiotic spindle. After oocyte activation, homologous chromosomes segregate between the oocyte and the first polar body, and a diploid pronucleus-like nucleus reforms from the one set of dyads. This nucleus condenses in the first embryonic mitosis into 40 ‘somatic’ chromosomes which coexist in the common metaphase plate with 20 somatic chromosomes originating from the female pronucleus. Shortening of the time between fusion and activation to about 1 h prevents bivalent differentiation. The PO nucleus condenses only partially and reforms, after oocyte activation, a pronucleus-like nucleus. This nucleus gives rise at the first embryonic mitosis to 20 bivalents which coexist with 20 somatic chromosomes originating from the female pronucleus. A PO nucleus introduced into an activated egg completes the first cell cycle as an intact interphase nucleus. It never condenses in the first embryonic mitosis into bivalents, and undergoes only initial condensation (preceding bivalent differentiation). These results indicate that: (1) condensation into bivalents, meiotic spindle formation and first meiotic division can be greatly accelerated by the introduction of an early diplotene (dictyate) oocyte nucleus into a metaphase II oocyte, and (2) depending on whether the diplotene nucleus enters the first embryonic (mitotic) cell cycle after just initiating or after completing the first meiosis, it gives rise at the first cleavage division to meiotic (bivalents) or ‘somatic’ chromosomes respectively.
Isolation of viable sperm cells from tobacco (Nicotiana tabacum)
Authors: Cao Yajuan , Reece Adonis , Russell Scott D. 
Published: September 2008

SummaryViable sperm cells of Nicotiana tabacum were isolated by the semi-vivo technique. After pollination, excised styles were floated, cut end immersed, in a solution of 15% sucrose with 0.01% boric and 0.03% Ca(NO3)2 at 27°C in a growth chamber until pollen tubes emerged. After sperm cells were formed (at least 8 h after pollination) tubes were immersed in a 9% mannitol solution. In this solution, sperm cells are nearly ellipsoidal and retain viability for over 6 h.
Mouse oocytes penetrated by sperm at GV or GVBD stage lose the ablity to fuse with additional spermatozoa
Authors: Pyrzyńska Beata , Maleszewski M. , Maluchnik D. 
Published: September 2008

SummaryMouse oocytes penetrated by spermatozoa during germinal vesicle (GV) breakdown undergo maturation and are arrested at metaphase of the second meiotic division despite the presence of sperm nuclei within the ooplasm. When these oocytes were re-inseminated, none was penetrated by newly added spermatozoa. When GV oocytes were inseminated and cultured in the presence of dibutyryl cAMP, the oocytes remained at GV stage, yet they did not permit entry of additional spermatozoa. These observations suggest that the plasma membrane of maturing oocytes is modified by precociously penetrating spermatozoa independently from cortical granule exocytosis. Sperm components incorporated into the oocytes seem to be responsible for the modification of the oocyte's plasma membrane.
Centrosomes with striated rootlets in rabbit zygotes
Authors: Szöllösi M.S. , Adenot P. , Szöllösi D. 
Published: September 2008

SummaryAn electron microscopic study of the rabbit zygote has shown the presence of numerous paracrystalline structures (PSs) around the pronuclei. The majority of these structures are situated in the narrow space between pronuclei. The PSs during interphase are associated with small dense knobs, and filamentous material;some of them, namely those situated in the internuclear space, are also associated with striated rootlets. The PS and its appendages form a complex which nucleates microtubules during interphase and phase M. The structure of these complexes changes with the cell cycle. Striated rootlets disappear at G2/M. Dense Knobs and filamentous material separate from the PS, become loose and associate with numerous microtubules at the poles of the first mitotic spindle. PSs and their associated structures are considered to be a newly discovered morphological form of the centrosome.
Structure and function of asterosaps, sperm-activating peptides from the jelly coat of starfish eggs
Authors: Nishigaki Takuya , Chiba Kazuyoshi , Miki Wataru , Hoshi Motonori 
Published: September 2008

SummaryJelly coat of starfish eggs has the capacity to activate homologous spermatozoa and induce the acrosome reaction. We have isolated 12 sperm-activating peptides (SAPs) from the egg jelly of the starfish,Asterias amurensis. Eleven SAPs were structurally identified by sequence analysis and electro-spray ionisation mass spectrometry. All of them are glutamine-rich tetratriacontapeptides with an intramolecular disulphide linkage between Cys8and Cys32. They are much larger than sea urchin SAPs and do not show any significant sequence similarities to known proteins. Thus we have collectively named them asterosaps. The amino terminal region, where structural diversity of asterosaps is observed, is not important for their activity, whereas the disulphide linkage is essential. Asterosaps do not induce the acrosome reaction by themselves, but are able to induce the acrosome reaction in combination with an egg jelly glycoconjugate named ARIS. Furthermore, anti-asterosap rabbit antibody significantly decreased the acrosome reaction-inducing activity of the jelly solution and the activity was restored by addition of excess asterosap. These results support our hypothesis that the main physiological role of SAPs is the induction of the acrosome reaction in cooperation with two other jelly components, ARIS and Co-ARIS.
Beneficial effect of medium with high concentration serum for direct sperm injection into mouse oocytes using a conventional pipette
Authors: Suzuki Kazuo , Yanagimachi R. 
Published: September 2008

SummaryAlthough the piezo-electrically driven pipette is much more effective than a conventional pipette for direct sperm injection into mouse oocytes, the piezo-pipette driving unit is expensive and may not be readily accessible to most basic laboratories. We reprot here that the inefficiency of the conventional pipette can be improved by performing the operation in a medium with high serum concentration.
Laser blastocyst biopsy for preimplantation diagnosis in the human
Authors: Veiga A. , Sandalinas M. , Benkhalifa M. , Boada M. , Carrera M. , Santaló J. , Barri P.N. , Ménézo Y. 
Published: September 2008

SummaryA new methodology for blastocyst biopsy that uses a 1.48μm in diode laser is described. Trophectoderm cells are biopsied after laster zona drilling and culture, fixed and processed for fluorescent in situ hybridisation (FISH) analysis. Preliminary results on the efficiency of the procedure and blastocyst recovery rate are promising. Blastocyst laser biopsy is a useful tool in preimplantation genetic diagnosis (PGD) as it allows a more reliable diagnosis and widens the diagnostic possibilities on account of the higher number of cells obtained in the biopsy.
Indicators and scientific knowledge in regional Baltic Sea environmental policy
Authors: Backer Hermanni 
Published: September 2008

Abstract
Backer, H. 2008. Indicators and scientific knowledge in regional Baltic Sea environmental policy. – ICES Journal of Marine Science, 65: 1398–1401. Since the 1990s, indicators have emerged as key tools used by the Helsinki Commission (HELCOM) for drafting regional Baltic Sea management actions and monitoring their implementation. The central role played by indicators in recent years can readily be seen in the 2007 Baltic Sea Action Plan (BSAP), where they have been used for defining quantitative targets and management actions and for providing background information. Similarly, the currently available, annually updated, web-based indicators include performance indicators with quantitative reference and target values, and descriptive indicators without such values. In addition to good indicators, implementation of the ecosystem approach, as is the aim of the BSAP as well as the newly adopted European Marine Strategy Framework Directive, requires extensive implementation of the precautionary principle and related concepts.