bluuebunny/crossref_metadata_2025 huggingface dataset:
Phonetic and phonological contrasts in the acquisition of voicing: voice onset time production in Hindi and English
Authors: Davis Katharine
Published: September 2008

The present study examines adult and child word-initial voice onset time productions in English and Hindi (10 adults and 20 children in each language) to determine the age of acquisition of the phonemic voice contrast. Cross-linguistic differences in patterns of acquisition were found, but these need not be traced to the different phonological Systemsper se. An examination of the data indicates that the best predictor of age of voice contrast acquisition across languages is one which rests on the actual acoustic differences between members of phonologically contrastive pairs. In general it was found that the larger the post-release voice onset time differences between pair members in the adult model, the earlier the contrast is reliably produced by child language learners.
Relative clauses are barriers to wh-movement for young children
Authors: Villiers Jill De , Roeper Thomas
Published: September 2008

Two studies are described which investigate preschool children's sensitivity to relative clauses as barriers to the movement of wh-questions. The children were presented with short stories followed by questions in which the wh-word had two possible sites of interpretation, the ungrammatical option being inside a relative clause. A cross-sectional study with 23 children aged 3;1 to 6;1, and a longitudinal study over the course of one year with 12 children aged 3;1 to 4;1 at the start, found young children refused to extract wh-questions from the ungrammatical site inside a relative clause. This confirms other findings that children's early grammars are sensitive to universal constraints on movement. In addition, the children differentiated between wh-complements and relative clauses in their tendency to mistakenly answer the medial wh-complementizer but not the wh-relative pronoun. Explanations for the latter are framed in terms of children's initial assumptions about the attachment of complements.
Functional changes in early child language: the appearance of references to the past and of explanations
Authors: Veneziano Edy , Sinclair Hermina
Published: September 2008

Spontaneous speech samples from children during the period of transition from one word to multi-word utterances in interaction with their French-speaking mothers were explored in order to study the appearance and development of functional changes in their use of language. Two types of such change were noted in the longitudinal records of four children when they were still essentially one-word speakers: the beginnings of references to the past, and the appearance of explanations and justifications, especially in communicative situations of request and refusal. The co-appearance of these behaviours is discussed in relation to two more general developmental changes: a detachment from the immediately perceptible situation linked to a further elaboration of the signifier-signified relation, and a socio-cognitive development leading to a view of the interlocutor as an alter ego, as a person whose psychological states may be different from the child's own.
Language differentiation in early bilingual development
Authors: Genesee Fred , Nicoladis Elena , Paradis Johanne
Published: September 2008

It has been claimed that children simultaneously acquiring two languages go through an initial stage when they are unable to differentiate between their two languages. Such claims have been based on the observation that at times virtually all bilingual children mix elements (e.g. lexical, morphological) from their two languages in the same utterance. That most, if not all, children acquiring two languages simultaneously mix linguistic elements in this way is widely documented. Although such code-mixing is not well understood or explained, there are a number of explanations unrelated to lack of language differentiation that may explain it. Moreover, while language differentiation is widely attested among bilingual children once functional categories emerge, usually during the third year, there is still some question as to how early in development differentiation is present. In this study, we examined language differentiation in five bilingual children prior to the emergence of functional categories (they ranged in age from 1;10 to 2;2 and in MLU from 1·23 to 2·08). They were observed with each parent separately and both together, on separate occasions. Our results indicate that while these children did code mix, they were clearly able to differentiate between their two languages. We also examine the possibility that the children's mixing is due to (a) their language dominance, and (b) their parents' rate of mixing. We could find no evidence that their mixing was due to parental input, but there was some evidence that language dominance played a role.
Conserving Mackinder's eagle owls in farmlands of Kenya: assessing the influence of pesticide use, tourism and local knowledge of owl habits in protecting a culturally loathed species
Authors: OGADA DARCY L. , KIBUTHU PAUL MURIITHI
Published: September 2008

Farmlands can be good habitat for owls and owls can benefit farmers, but pesticide use can negatively affect owls and within many regions of Africa owls are loathed owing to beliefs that they bring misfortune or death. Since 1997, a small-scale owl tourism initiative that educates farmers about owls and benefits them financially has been operating in central Kenya. Pesticide use, farmers' beliefs and knowledge about owls, and the impacts that tourism can have on farmers' attitudes and behaviour towards owls in rural Kenya were surveyed. Agricultural pests were the most serious problem facing farmers, though only 28 % of farmers said they controlled vertebrate pests using pesticides. The insecticide carbofuran was often misused to kill vertebrate pests. Common control measures were either to ‘do nothing’ or to chase pests from farms. Farmers knew of Mackinder's eagle owls living adjacent to their farms, but 68 % said they didn't adhere to the culturally negative beliefs about owls. Knowledge of owl diet amongst farmers was high (75 %). Farmers who benefited from owl tourism were more likely to know more about owl diet and habits. Where farmers gained financial benefits from tourism or knew more about owls, they were more likely to categorize owls as ‘good’, but farmers who knew about owl diet were more likely to use pesticides or kill owl prey on their farms. Though knowledge of owls did not have a positive effect on farmers' behaviour toward owls, this was probably the result of a lack of ecological literacy rather than any deliberate antagonism toward owls. Financial rewards are very important to poor farmers, but may not result in actions that enhance species conservation unless farmers have a basic understanding of ecological processes.
Sojourn time distribution in some processor-shared queues
Authors: Tan Xiaoming , Knessl Charles
Published: September 2008

We develop a technique for obtaining asymptotic properties of the sojourn time distribution in processor-sharing queues. We treat the standard M/M/1-PS queue and its finite capacity version, the M/M/1/K-PS queue. Using perturbation methods, we construct asymptotic expansions for the distribution of a tagged customer's sojourn time, conditioned on that customer's total required service. The asymptotic limit assumes that (i) the traffic intensity is close to one for the infinite capacity model, and (ii) that the system's capacity is large for the finite capacity queue.
A simple model of the melt fracture instability
Authors: Greenberg J. M. , Demay Y.
Published: September 2008

In this note we develop and analyse a system of equations describing a molten linear polymer being extruded in a capillary rheometer which is operating under the controlled condition that the mean velocity at the capillary inlet is maintained at a constant value. Slipping of the melt at the pipe wall is permitted and an evolution equation for the boundary slip parameter is postulated. The combined system governing the mean cross-sectional velocity and slip parameter is shown to exhibit relaxation oscillations similar to those observed in actual rheometers.
Self-similar solutions of the second kind for the modified porous medium equation
Authors: Hulshof Josephus , Vazquez Juan Luis
Published: September 2008

We construct compactly supported self-similar solutions of the modified porous medium equation (MPME)

They have the form

where the similarity exponents α and β depend on ε, m and the dimension N. This corresponds to what is known in the literature as anomalous exponents or self-similarity of the second kind, a not completely understood phenomenon. This paper performs a detailed study of the properties of the anomalous exponents of the MPME.
Nucleation of superconductivity in decreasing fields. II
Authors: Chapman S. J.
Published: September 2008

The bifurcation from a normally conducting to a superconducting state as an external magnetic field is lowered is examined using the Ginzburg–Landau theory. Linear and weakly nonlinear stability analyses are performed near the bifurcation point, and the implications of the results for each of three examples is considered.
Numerical optimization and quasiconvexity
Authors: Gremaud P. A.
Published: September 2008

In the Calculus of Variations, several notions of convexity have emerged, corresponding to different properties of the functionals to be minimized. The relations between these various notions are not yet fully understood. In this context, we present a numerical study of quasiconvexity for some functions of the type f(ξ) = g(|ξ|2, det ξ), where Ξ is a 2×2-matrix. The corresponding global optimization problems are solved using a simulated annealing-like algorithm. The computations strongly indicate that the considered functions are quasiconvex if and only if they are rank-one convex. The relation to Morrey's conjecture, various applications and implementation problems are discussed.
Cusp development in free boundaries, and two-dimensional slow viscous flows
Authors: Howison S. D. , Richardson S.
Published: September 2008

We consider a family of problems involving two-dimensional Stokes flows with a time dependent free boundary for which exact analytic solutions can be found; the fluid initially occupies some bounded, simply-connected domain and is withdrawn from a fixed point within that domain. If we suppose there to be no surface tension acting, we find that cusps develop in the free surface before all the fluid has been extracted, and the mathematical solution ceases to be physically relevant after these have appeared. However, if we include a non-zero surface tension in the theory, no matter how small this may be, the cusp development is inhibited and the solution allows all the fluid to be removed.
Theorem on the existence of solutions of quasi-static moving boundary problems
Authors: Obbink Bart Klein
Published: September 2008

Using the theory of conformal mappings, we show that two-dimensional quasi-static moving boundary problems can be described by a non-linear Löwner-Kufarev equation and a functional relation ℱ between the shape of the boundary and the velocity at the boundary. Together with the initial data, this leads to an initial value problem. Assuming that ℱ satisfies certain conditions, we prove a theorem stating that this initial value problem has a local solution in time. The proof is based on some straightforward estimates on solutions of Löwner-Kufarev equations and an iteration technique.
Convergence of a numerical method in mathematical spontaneous potential well-logging
Authors: Zhou Yi , Cai Zhijie
Published: September 2008

Spontaneous potential well-logging is an important technique in petroleum exploitation. The spontaneous potential satisfies an elliptic boundary value problem with jump conditions on interfaces. At the joint points of the interfaces, the jumps of the spontaneous potential do not, in general, satisfy the compatibility condition. It turns out that it is impossible to find a piecewiseH1solution to the problem, and the standard finite element method cannot be applied to get an approximate solution. In this paper, by means of a new method, we prove that the problem exists a unique piecewisesolution for somep< 2. We give an estimate for thesolution as well. This allows us to prove the convergence of a numerical scheme proposed in [3].
On the solution of a class of polydisperse spray problems
Authors: Anidjar F. , Greenberg J. B. , Tambour Y.
Published: September 2008

A general method is presented to facilitate the solution of a class of polydisperse spray problems in which a cloud of droplets can be described using a sectional or group model. The procedure involves replacing the originalcoupleddroplet sectional variables conservation equations by a set ofuncoupledsectional equations for auxiliary variables. The form of these latter equations is identical to that of thesinglespray equation for a quasi-monodisperse spray, solutions of which are more readily attainable even for multidimensional spray problems. Thus, these ready-made solutions can be exploited directly for the auxiliary variables, from which solutions can then be constructed in a straightforward manner for the desired original sectional variables. Three illustrative examples for spray diffusion flames with different features of complexity highlight the potential applicability of the proposed method, and indicate the sensitivity of flame characteristics to initial spray conditions and in-spray related phenomena.
A mean-field model of superconducting vortices
Authors: Chapman S. J. , Rubinstein J. , Schatzman M.
Published: September 2008

A mean-field model for the motion of rectilinear vortices in the mixed state of a type-II superconductor is formulated. Steady-state solutions for some simple geometries are examined, and a local existence result is proved for an arbitrary smooth geometry. Finally, a variational formulation of the steady-state problem is given which shows the solution to be unique.
On the Mróz model
Authors: Brokate Martin , Dressler Klaus , Krejčí Pavel
Published: September 2008

We treat the mathematical properties of the one-parameter version of the Mróz model for plastic flow. We present continuity results and an energy inequality for the hardening rule, and discuss different versions of the flow rule regarding their relation to the basic laws of thermodynamics.
Mathematical modelling of the motion of hard contact lenses
Authors: Moriarty J. A. , Terrill E. L.
Published: September 2008

In this paper we examine the movement of hard contact lenses on the eye. In so doing, we take into account hydrodynamic forces underneath the lens, as well as surface tension forces at the lens periphery. This involves solving for the free surface of the tear film away from the lens in order to determine the magnitudes of the pressure and surface tension forces on the lens. The analysis, which assumes quasi-steady motion, is carried out in both two and three dimensions.
Asymptotic profiles of a decaying contaminant in transport through a porous medium
Authors: Escobedo M. , Grundy R. E.
Published: September 2008

M. Escobedo and R. E. Grundy Vol. 7 (1996), pp. 395–416; The publisher apologises for an oversight which resulted in an error in the above paper. The following three figures were inadvertently published in an incomplete form, with some of the labelling omitted.
Cloning, sequencing and oocyte-specific expression of the marmoset sperm receptor protein, ZP3
Authors: Thillai-Koothan P , van Duin Marcel , Aitken R. John
Published: September 2008

The zona pellucida surrounding the mammalian oocyte contains a major glycoprotein species, ZP3, that serves as a cell- and species-specific receptor for spermatozoa. In this study we have determined the primary amino acid structure of marmoset ZP3 (marZP3) and examined the expression of marZP3 mRNA within the ovary. The marZP3 gene possesses an open reading frame of 1272 nucleotides which is expressed specifically by the oocyte and encodes a polypeptide chain of 424 amino acids that exhibits 91% homology with the human ZP3 sequence. The disparity between these molecules was confined to a short domain spanning residues 322–352; otherwise the molecules were very similar, showing conservation of many structural features including theN–linked glycosylation sites, location and number of cysteine and proline residues and hydrophobicity profile. The results of this study have important implications for the use of the marmoset monkey as an animal model for the development of contraceptive vaccines targeting ZP3.
Vitrification of rat blastocysts developed in vitro
Authors: Nakamichi R , Ohboshi S , Fujihara N
Published: September 2008

Approximately 50% of rat 8-cell embryos obtained from the oviduct on day 4 of pregnancy developed to the balstocyst stage after 18 h of incubation in vitro. The embroys were compared with in vivo blastocysts derived from the uterus on day 5 of pregnancy as regards their response to vitrification treatment. Before vitrification, both types of embryos were exposed to vitrification solution, and subsequent embryonic development was inhibited with increasing time of exposure. A greater suppression of development after exposure was observed in the embryos cultured in vitro compared with in vivo blastocysts. However, development was gradually restored as in vitro incubation was continued. The response of embryos to vitrification and warming treatments was shown to be almost the same for both in vitro and in vivo blastocysts, though the former were significantly (p < 0.05) less tolerant of exposure to vitrification solution. These results suggest that in vitro blastocysts were much more susceptible to vitrification solution than in vivo blastocysts. The degree of damage caused by vitrification and thawing treatments, and the inhibition of in vitro embryonic development, were significantly (p < 0.05) more serious for in vitro blastocysts.
A rho-like protein is involved in the organisation of the contractile ring in dividing sand dollar eggs
Authors: Mabuchi Issei , Hamaguchi Yukihisa , Fujimoto Hirotaka , Morii Narito , Mishima Masanori , Narumiya Shuh
Published: September 2008

Sand dollar eggs were microinjected with botulinum C3 exoenzyme, an ADP-ribosyltransferase fromClostridium botulinumthat specifically ADP-ribosylates and inactivates rho proteins. C3 exoenzyme microinjected during nuclear division interfered with subsequent cleavage furrow formation. No actin filaments were detected in the equatorial cortical layer of these eggs by rhodamine-phalloidin staining. When microinjected into furrowing eggs, C3 exoenzyme rapidly disrupted the contractile ring actin filaments and caused regression of the clevage furrows. C3 exoenzyme had no apparent effect on nuclear division, however, and multinucleated embryos developed from the microinjected eggs. By contrast, C3 exoenzyme did not affect the organisation of cortical actin filaments immediately after fertilisation. Only one protein (molecular weight 22000) was ADP-ribosylated by C3 exoenzyme in the isolated cleavage furrow. This protein co-migrated with ADP-ribosylated rhoA derived from human paltelets when analysed by two-dimensional gel electrophoresis. These results strongly suggest that a rho-like, small GTP-binding protein is selectively in the organisation and maintenance of the contractile ring.
Ultrastructural studies of early mouse embryos obtained by oocyte fusion
Authors: Nogués Carme , ponsà Montserrat , Egozcue Josep , Vidal Francesa
Published: September 2008

Oocyte fusion induced by inactivated Sendai virus results in the production of ‘zygotes’that are able to undergo the first stages of embryonic development. The oocyte fusion products (OFP) obtained follow a morphological developmental pattern equivalent to that of control embryos, at least up to the 8-cell stage. The percentage of OFP that reach the 8-cell stage is extremely low(3%)compared with control embryos culturedin vitro(95%). On light microscopy, the OFP obtained show morphological characteristics identical to control embryos, although their cell diameters are larger. The cortical reaction, meiotic reactivation, extrusion of second polar bodies and pronucleus formation take place as observed in controls. The ultrastructural characteristics of oocyte fusion products at the 1-2-4- and 8- cell stages are analogous to those of controls, including the presence of structures related to the activation of the embryo genome. However some differences concerning cell ultrastructure, mainly in the nucleus, are observed and discussed in the text.
Isolation and microinjection of active sperm nuclei into egg cells and central cells of isolated maize embryo sacs
Authors: Matthys-Rochon E. , Mòl R. , Heizmann P. , Dumas C.
Published: September 2008

Artificial fertilisation was attempted in maize by microinjecting sperm nuclei into the egg cell or central cell of isolated embryo sacs. A protocol for isolation of nuclei from pollen grains was developed and a pure fraction of sperm nuclei was obtained after centrifugation on a Percoll gradient. The in vitro transcriptional activity of the nuclei was tested by incorporation of radioactive UTP into RNA. The level of labelled nucleotide incorporation increased and reached a maximum after between 30 and 40 min in the incubation medium. The embryo sacs were enzymatically isolated and their viability determined by observation of cytoplasmic streaming in the female cells. The embryo sacs were immobilised by embedding in low-melting-point agarose and a single male nucleus was injected with a bevelled microcapillary. The presence of the injected nucleus in the egg or central cell was demonstrated using a cytological approach. This paper presents an alternative method for studying the intimate processes of fertilisation in plants.
Mammalian follicle-stimulating hormone stimulates DNA synthesis in secondary spermatogonia and Sertoli cells in organ culture of testes fragments from the newt,Cynops pyrrhogaster
Authors: Ji Zai Si , Abeé Shin-Ichi
Published: September 2008

We previously showed in organ culture of testes fragments fromCynops pyrrhogasterthat mammalian folicle-stimulating hormone (FSH) stimulates secondary spermatogonia to differentiate into primary spermatocytes. In this report, we demonstrate in organ culture that FSH stimulates DNA synthesis in secondary spermatogonia and Sertoli cells: the numbers of secondary spermatogonia and Sertoli cells incorporating 5-bromo-2′ -deoxyuridine (BrdU)throughout the culture period in the presence of FSH were 3-5 times those incorporating BrdU in the absence of FSH. Moreover, addition of FSH, induced after a day a remarkable increase in the number of spermatogonia and Sertoli cells incorporating BrdU. The above results indicate that FSH stimulates and induces DNA synthesis in spermatogonia and Sertoli cells. Most of the spermatogonia within a cyst were labelled simultaneously and at the same density, indicating that they underwent synchronous DNA synthesis, whereas all the Sertoli cells within a cyst were not labelled simultaneously, indicating that they synthesised DNA asynchronously. When testes fragments pulse-labelled with Brdu were cultured in FSH for 14 days, the secondary spermatogonia differentiated into primary spermatocytes, whereas in the absence of FSH they failed to differentiate and most died by the 7th day. The above results together show that FSH is required for the proliferation of both secondary spermatogonia and Sertoli cells as well as the differentiation of secondary spermatogonia into primary spermatocytes.
Confocal microscopy of F-actin distribution inXenopusoocytes
Authors: Roeder Amy D. , Gard David L.
Published: September 2008

We have used rhodamine-conjugated phalloidin and confocal microscopy to examine the organisation of filamentous actin (F-actin) during oogenesis inXenopus laevis. F-actin was restricted to a thin shell in the cortex of oogonia and post-mitotic oocytes less than 35 μm in diameter. In oocytes with diameters of 35–50 μm F-actin was observed in three cellular domains: in the cortex, in the germinal vesicle (GV) and in a network of cytoplasmic cables. Initially, actin cables were sparsely distributed in the cytoplasm, with no evidence of discrete organising centres. In larger stage I oocytes, a dense network of actin cables extended throughout the cytoplasm, linking the GV and mitochondrial mass to the cortical actin shell. Apart from the F-actin associated with the mitochondrial mass, no evidence of a polarised distribution of F-actin was apparent in stage I oocytes. F-actin was observed also in the cortex and the GV of stage VI oocytes, and a network of cytoplasmic cables surrounded the GV. Cytoplasmic actin cables extended from the GV to the animal cortex, and formed a three-dimensional network surrounding clusters of yolk platelets in the vegetal cytoplasm. Finally, disruption of F-actin in stage VI oocytes with cytochalasin resulted in distortion and apparent rotation of the GV in the animal hemisphere, suggesting that actin plays a role in maintaining the polarised organisation of amphibian oocytes.
Sperm entry into fertilised mouse eggs
Authors: Maluchnik Darek , Borsuk Ewa
Published: September 2008

We have investigated the oolemma block to polyspermy in the mouse. Zona-free and zona-intact eggs were fertilised and subsequently re-inseminated (the latter following zona pellucida removal). The ‘perivitelline’ block to polyspermy in zona-intact eggs renders motile sperm in the perivitelline space unable to bind to the oolemma. This is not connected with irreversible changes in the egg plasma membrane, because freshly added sperm can still fuse with such eggs freed from the zona. Fertilised eggs eventually lose the ability to fuse with sperm within 1 h, while still being able to bind many sperm.
Producing exposed cota-free embryos
Authors: Daily Michael F. , Latham Virginia H. , Garcia Claudia M. , Hockman Cynthia L. , Chun Helen , Oppenheimer Mark L. , West Steven P. , Rostamiany Karolin , Chao Richard L.C. , Pollock Edward G. , Oppenheimer Steven B.
Published: September 2008

Production of embryos that are free of tough outer coats facilitates studies that are not possible with embryos surrounded by impenetrable envelopes. This report describes a new procedure for preventing formation of fertilisation membranes in the sea urchin (Lytechinus pictus) model. This procedure involves treating unfertilised eggs with the enzyme alpha-amylase, which cleaves alpha-1,4 glucosidic bonds in the vitelline layer. A major advantage of this method is that it is very well defined and completely controllable with alpha-amylase inhibitor. The results suggest that intact alpha-1,4 glucosidic bonds are essential for vitelline layer integrity required for formation of the fertilisation membrane. Eggs treated with alpha-amylase possessed the same surface lectin receptors as untreated eggs and, as shown by light and transmission electron microscopy, produced healthy, cleaving embryos that were free of fertilisation envelopes.
Plasminogen activatior inhibitor type 1 in rat spermatozoa: localisation in the tail, in the acrosome and on the surface of the head
Authors: Manske Marianne , Welte Beate , Hitschke Karen , Wack Andreas , Bade Ernesto G.
Published: September 2008

Isolated seminiferous tubules of rat testis contain considerable urokinase-inhibiting activity. An immunohistological analysis revealed the presence of plasminogen activator inhibitor type 1 (PAI-1) in the basement membrane as well as in the interior of the tubules. Distribution and intensity of the intratubular immunoreactivity depends on the tubules. Distribution and intensity of the intratubular immunoreactivity depends on the stage of the seminiferous cycle. A relatively weak signal is present around elongated nuclei of spermatids at the beginning of chromatin condensation. The signal intensity increases in the course of differentiation until a maximum is reached at stages VII-VIII. In these stages PAI-1 immunoreactivity is localised around the nuclei of the late spermatids as well as along their tails. Spermatozoa in the ductus epididymis also strongly react with the PAI-1-specific antiserum, suggesting that the inhibitor remains associated with the germ cells after spermiation and during maturation in the epididymis. In intact mature spermatozoa isolated from epididymis cauda by ‘swimming-up’ in non-capacitation medium, PAI-1 antigen is localised on the plasma membrane surrounding the head. In addition, in fixed and permeabilised cells the immunoreactivity is detectable in the acrosome and in the tail. Possible functions of PAI-1 in spermatogenesis, sperm motility and sperm-egg interaction are discussed.
Inhibition of sperm-egg fusion in the hamster and mouse by carbohydrates
Authors: Ponce Ruben H. , Urch Umbert A. , Yanagimachi Ryuzo
Published: September 2008

After spermatozoa bind to and penetrate the extracellular matrix of the egg, the zona pellucida, they adhere to and fuse with the plasma membrane of the egg. Since sperm–egg fusion may involve membrane glycoproteins and/or carbohydrate binding proteins, we sought to test this hypothesis by challenging sperm–egg fusion in hamster and in mouse with added carbohydrates. In this study, a number of carbohydrate and glycoconjugates were examined for their ability to inhibit sperm–eggfusion. In the hamster, D(+)-glucosamine, D(+)-galactosamine, albumin-bovine-glucosamide and-galactosamide, fucoidan and dextran sulphate inhibited the fusion of spermatozoa with zona-free eggs. The same effects were seen in the mouse, except for the toxic effects of D(+)-galactosamine. These facts suggest a role of carbohydrate binding proteins or glycoproteins in the fertilisation process at the level of binding to and fusing with the oolemma.
Transcription of paternal Y-linked genes in the human zygote as early as the pronucleate stage
Authors: Ao Asangla , Erickson Robert P. , Winston Robert M.L. , Handysude Alan H
Published: September 2008

Global activation of the embryonic genome occurs at the 4– to 8–cell stage in human embryos and is marked by continuation of early cleavage divisions in the presence of transcriptional inhibitors. Here we demonstrate, using recerse transcripase–polymerase chin reaction (Rt–PCR), the presence of transcripts for wo paternal Y chromosomal genes,ZFYandSRYin human preimplantation embryos.ZFYtranscripts were detected as early as the pronucleate stage, 20–24 h post-inseminationIn vitroand at intermediate stages up to the blastocyst stage.SRYTranscripts were also detected at 2–cell to blastocyos observed in many mammalian species focuses attention on the role of events in six determination prior to gonad differentiation.
Cell–cell interaction and oocyte growth
Authors: Canipari Rita
Published: September 2008

In most mammals, oocytes initiate meiosis in late fetal life; by the time of birthe they have already entered the diplotene stage of prophase I of meiosis and becaome arrested thereafter at the dictyate state(Baker, 1972). At this stage they became surrounded by a few nonproliferating flat follicle cells forming a unit called the resting or primordial follicle.
Ovarian regulation of sperm progression in the Fallopain tubes
Authors: Hunter R. H. F.
Published: September 2008

Successful liberation and apposition of gametes are essential components of fertility. Normal fertilisation depends upon the establishment of a pre-ovulatory sperm gradient in the female tract between the site of semen deposition and the site of fertilisation in the Fallopian tubes. As a consequence, sperm: egg ratios may be close to unity at the time of activation of most secondary oocytes under conditions of spontaneous mating. In the absence of a sufficient sperm gradient, newly ovulated eggs would be confronted by an excess of spermatozoa resulting in polyspermic fertilisation. Penetration of the vitellus by more than one spermatozoon is pathological in mammals (Beatty, 1957; Austin, 1963). Accordingly, systems that act to regulate sperm progression and competence before the time of ovulation assume a particular importance. During the 1950s, 1960s and 1970s, there was much controversy as to the rate of sperm transport into the Fallopian tubes. Because observations failed to focus on those spermatozoa that could fertilise eggs, the controversy was largely sterile. Nor were the disagreements well founded since some experiments employed artificial insemination whilst others used natural mating. These two quite distinct approaches to introducing a sperm suspension into the female tract could not reasonably form the basis of disagreements on the physiological events of cellular progression. More recent studies have been set in perspective by Overstreet (1983), Harper (1988), Yanagimachi (1988), Hunter (1988, 1991, 1995) and Drobnis & Overstreet (1992)After mating at the onset of oestrus, ram and bull spermatozoa require a minimum of 6–8 h to reach the Fallopian tubes in sufficient numbers to promote suc.cessful fertilisation (Hunter et al., 1980; Hunter & Wilmut, 1982). Spermatozoa displaced to the tubes in a small number of minutes are moribund or dead, not.
Expression of the cell cycle control protein cdc25 in cleavage stage bovine embryos
Authors: Jones J.M. , First N.L.
Published: September 2008

We have examined the synthesis and expression of a homologue of the cell cycle protein cdc25 by early cleavage stage bovine embryos. cdc25 is the protein phosphatase responsible for activating p34cdc2 by dephosphorylating the threonine 14 (Thr 14) and tyrosine 15 (Tyr 15) residues of p34cdc2. Human cdc25 antibody was utilised in western blots immunoprecipitations to examine the presence and synthesis of cdc25 in bovine embryos. cdc25 is present as a 52 kDa non-phosphorylated and a 66 kDa presumably phosphorylated form in bovine 1−, 2−, 4− and 8−cell embryos. However, cdc25 is actively synthesised only in 8-Cell embryos, indicating that the cdc25 present prior to this stage is inherited from the oocyte. In addition, the synthesis of cdc25 was induced in 2-cell embryos in which cleavage was blocked with the DNA synthesis inhibitor aphidicolin.
PCR-generated cDNA libraries from reduced numbers of mouse oocytes
Authors: Revel Frédérique , Renard Jean-Paul , Duranthon Véronique
Published: September 2008

We describe a rapid and reproducible method for cloning cDNA amplified from 10 mouse oocytes. The procedure consists in priming cDNA synthesis from a crude cellular extract using an oligo d(T) containing primer and submitting the size-limited cDNA first strand to poly(dG) tailing. The whole cDNA population is then polymerase chain reaction (PCR) amplified using two primers complementary to oligo d(A) and oligo d(G) ends of the cDNA. In this procedure no purification steps are required. We obtained about 5 ×106clones from 10 oocytes. Screening of the library showed that the relative abundance of the transcripts was preserved during amplification and cloning and that the procedure allows cloning of low-abundance sequences at least as rare as 0.008% of the mRNA. The repeatable generation of representative cDNA libraries from reduced numbers of oocytes or embryos should open new opportunities for obtaining genetic information from mammalian preimplantation embryos.
Activation of mitogen-activated protein kinase during meiotic maturation in porcine oocytes
Authors: Inoue Maki , Naito Kunihiko , Aoki Fugaku , Toyoda Yutaka , Sato Eimei
Published: September 2008

To investigate the involvement of mitogen-activated protein kinase(MAP kinase) in meiotic maturation of porcine oocytes, we assayed MAP kinase activity using basic protein(MBP) as a substrate. MAP kinase activity was low during the germinal vesicle stage, 0–20 h of culture. An abrupt increase was observed at metaphase I(30 h of culture), and activity remained significantly higher than that at 0 h until 50 h of culture, with a transient slight decrease at the time of first polar body extrusion (40 h). Detection of the kinase activity by an in-gel phosphorylation assay confirmed that the 42 and 44 kDa MAP kinases were significantly activated in 45 h cultured oocytes but not in 0 h oocytes, and just slightly in 20 h oocytes. In immunoblotting, however, the 42 and 44 kDa bands were detected in 0, 20 and 45 h cultured oocytes. Furthermore, the signal strength of the two bands did not change during the period of culture, but shifted up to 45 h, indicating that the activation of MAP kinase depended not on the synthesis but on the phosphorylation of this enzyme. These results suggest that the activation of MAP kinase is involved in the regulation of meiotic maturation of porcine oocytes, and especially in the regulation after germinal vesicle breakdown.
Response of avian nuclei to mammalian maturation promoting factor (MPF)
Authors: Trefil P. , Horská M. , Kaňka J. , Fulka J. , Jirmanová J. , Moor R.M. , Fulka J
Published: September 2008

Chicken blastodermal cells (stage X) were fused to mouse enucleated oocytes with either no or high maturation promoting factor (MPF) activity. High MPF levels always induced premature chromosome condensation (PCC) irrespective of the number of nuclei fused to a single oocyte. When a single blastodermal cell was fused to a single oocyte without MPF activity the nucleus remained intact for up to 3 h and thereafter underwent PCC. A quite different situation was observed after multiple fusion of several blastodermal cells to a single oocyte without MPF activity. Here, the transplanted nuclei remained intact even after prolonged culture but underwent extensive swelling. DNA synthesis was detected in almost all unfused blastodermal cells. However, after the fusion of several blastodermal cells to a single oocyte no DNA synthesis could be detected. These results provide further evidence that MPF is the universal cell-cycle regulator in the animal kingdom. Its activity is blocked (or neutralised) after fusion to several S-phase cells. Interestingly, our results further suggest that DNA synthesis is suppressed in meiotic cytoplasm even in the presence of an intact nuclear envelope.
Immunocytochemical localisation of the nucleolar protein fibrillarin and RNA polymerase I during mouse early embryogenesis
Authors: Cuadros-Fernández Jorge M. , Esponda Pedro
Published: September 2008

We have employed immunocytochemical procedures to localise the nucleolar protein fibrillarin and the enzyme RNA polymerase I in the numerous dense fibrillar bodies (nucleolar precursor bodies) which appear in the nuclei of mammalian early embryos. The aim of this study was to search for relationships between the localisation of these proteins, the changes in the structure of the nucleolar precursor bodies and the resumption of rRNA gene transcription during mouse early embryogenesis. Three human autoimmune sera which recognised fibrillarin and a rabbit antiserum created against RNA Polymerase. I were employed for fluorescence and electron microscopic immunocytochemical assays. A statistical analysis was also applied. Immunocytochemistry revealed that fibrillarin and RNA polymerase I showed the same localisation in the nucleolar precursor bodies. These proteins were immunolocalised only from the late 2-cell stage onward. Fibrillarin was initially detected at the periphery of the nucleolar pricursor bodies and the labelling gradually increased until the morula and blastocyst stages, where normally active nucleoli are found. The pattern of increase of fibrillarin during early embryogenesis shows a parallelism with the rise in rRNA gene transcription occurring during these embryonic stages, and a possible correlation between these two phenomena is suggested. Results demonstrated that nucleolar precursor bodies differ in their biochemical composition from the nucleolus and also from the prenucleolar bodies which appear during mitosis. When anti-fibrillarin antibodies were microinjected into the male pronucleus of mouse embryos to analyse the functions of fibrillarin during early development, they partially blocked the early development of mouse embryos and only 23.8% of injected embryos reach the blastocyst stage.
Microfilament assembly and cortical granule distribution during maturation, parthenogenetic activation and fertilisation in the porcine oocyte
Authors: Kim Nam-Hyung , Day Billy N. , Lee Hoon Taek , Chung Kil-Saeng
Published: September 2008

In this study we imaged integral changes in microfilament assembly and cortical granule distribution, and examined effects of microfilament inhibitor on the cortical granule distribution during oocyte maturation, parthenogenetic activation andin vitrofertilisation in the pig. The microfilament assembly and cortical granule distribution were imaged with fluorescent-labelled lectin and rhodamine-labelled phalloidin under laser scanning confocal microscopy. At the germinal vesicle stage, cortical granule organelles were located around the cell cortex and were present as a relatively wide area on the oolemma. Microfilaments were also observed in a wide uniform area around the cell cortex. Following germinal vesicle breakdown, microfilaments concentrated in the condensed chromatin and cortical granules were observed in the cortex. Treatment with cytochalasin B inhibited microfilament polymerisation and prevented movement of cortical granules to the cortex. Cortical granule exudation following sperm penetration was evenly distributed in the entire perivitelline space. These results suggest that the microfilament assembly is involved in the distribution, movement and exocytosis of cortical granules during maturation and fertilisation.
Comparison of method for activating mouse oocytes for spermatid nucleus tranfer
Authors: Sasagawa Isoji , Yanagimachi R.
Published: September 2008

In the mouse, mature oocytes injected with prespermatozoal cell nuclei remain unactivated. Additional stimulation is needed to trigger oocyte activation leading to embryo development. We compared various electrical stimulations, treatment with cycloheximide alone or in combination with electrical stimulation, and injection of sperm-borne oocyte-activating factor (oscillogen) in terms of their oocyte activation and embryo development rates. Of all the treatments tested, a single electrical pulse (1.0 kV / cm, 128 μs) was the simplest, yet very effective, in allowing the development of the oocytes injected with spermatid nuclei.
Cumulus expansion initiates physical and developmental autonomy of the oocyte
Authors: Larsen William J. , Chen Lin , Powers Robert , Zhang Hong , Russell Paul T. , Chambers Carolyn , Hess Karla , Flick Robert
Published: September 2008

As meiosis is initiated and the oogonium is transformed into a primary oocyte, the female germ cell becomes intimately invested by a single squamous layer of sex cord epithelium. As the follicle cell population expands during the initial stages of the ovarian cycle, oocyte and follicle cells become increasingly connected to one another by one of the most extensive populations of gap junctions documented in any epithelium (reviewed in Larsen & Wert, 1988).
Cyclopiazonic acid induces accelerated progress of meiosis in pig oocytes
Authors: Petr Jaroslav , Rozinek Jirří , Jílek František
Published: September 2008

In mammalian oocytes, calcium plays an important role in the regulation of meiotic maturation. In our study, we used the mycotoxin cyclopiazonic acid (CPA), an inhibitor of calcium-dependent ATPases, to mobilise intracellular calcium deposits duringin vitromaturation of pig oocytes. The CPA treatment of maturing oocytes significantly accelerated the progress of their maturation. Oocytes entered the CPA-sensitive period after 21 h ofin vitroculture. A very short (5 min) exposure to CPA (100 mM) is sufficient to accelerate maturation and it seems that accelerated maturation can be triggered by a transient elevation of intracellular calcium levels. The effect of CPA is not mediated through the cumulus cells, because maturation is accelerated by CPA treatment even in oocytes devoid of cumulus cells. Culture of oocytes with the calcium channel blocker verapamil (concentrations ranging from 0.01 to 0.04 mM) blocked the progress of oocyte maturation beyond the stage of metaphase I. This block can be overcome by the mobilisation of intracellular calcium deposits after CPA treatment (100 nM). The microinjection of heparin (20 pl, 50.1 mg/;ml), the inhibitor of inositol triphosphate receptors, before CPA treatment prevented the acceleration of oocyte maturation. This indicates that CPA mobilises the release of calcium deposits through inositol trisphosphate receptors. On the other hand, the microinjection of procaine (20 pl, 200 nM) or the microinjection of ruthenium red (20 pl, 50 mM), both inhibitors of ryanodine receptors, did not prevent accelerated maturation in CPA-treated oocytes. If present in pig oocytes, ryanodine receptors evidently play no part in the liberation of calcium from intracellular stores after CPA treatment.
Polyspermy in Bufo arenarum oocytes matured in vitro
Authors: Oterino J. , Toranzo G. Sánchez , Zelarayán L. , Bühler M.I.
Published: September 2008

Full-grown ovarian oocytes of the amphibian Bufo arenarum were induced to mature in vitro by removing the follicular layers (spontaneous maturation) or by treatment with progesterone (hormone-induced maturation). These oocytes were then treated with trypsin and inseminated with homologous spermatozoa. Oocytes matured in vivo that had not undergone any influence of the oviducts (coelomic oocytes), inseminated under the same experimental conditions, were used as controls. The results show that oocytes induced to mature in vitro and exhibiting apparently normal signs of activation were polyspermic. In fact, 2 h after insemination numerous functioning pronuclei could be observed in the animal hemisphere. These results suggest that even though the oocytes which matured in vitro were able to undergo activation after insemination, they were unable to establish an effective block to polyspermy.
Strain difference in the timing of meiosis resumption in mouse oocytes: involvement of a cytoplasmic factor(s) acting presumably upstream of the dephosphorylation of p34cdc2kinase
Authors: Polanński Zbigniew
Published: September 2008

Oocytes from eight inbred strains of mice were screened for the timing of germinal vesicle breakdown (GVB)in vitro. This characteristic varied between strains, reaching most extreme values in oocytes from AKR and BALB/c mice (3.1 and 1.6h after release from dibutyryl cAMP block, respectively;p<0.0001). The difference between AKR and BALB/c mice was confirmed in experiments in which GVB was inducedin vivoby stimulation with exogenous gonadotrophins. Analysis of the rate of GVB in hybrids obtained after fusion of nuclear and cytoplamic fragments of oocytes from both strains suggests that the factor responsible for the difference between AKR and BALB/c mice is located in the cytoplasm of the proghase oocytes. Finally, in oocytes from both strains stimulated to resume meiotic maturation with okadaic acid, an inhibitor of protein phosphatases types 1 and 2A the rate of GVB was the same (2.2h and 2.3h for AKR and BALB/c, respectively;p= 0.48). This suggests that the difference between strains is not related to the amount or quality of the pre-MPF (Maturation Promoting Factor) stored in the prophase oocyte, but to the factor(s) acting upstream of the dephosphorylation ofp34cdc2. kinase in the pathway leading to pre-MPF activation.