bluuebunny/crossref_metadata_2025 huggingface dataset:
Second messenger signalling during hormone-inducedXenopusoocyte maturation
Authors: Cork R. John , Robinson Kenneth R.
Published: September 2008

Although much information about such processes as cell cycle control, second messenger systems, protein kinases and steroid hormone action has been collected from studies ofXenopusoocyte maturation, we still have very little idea about how the steroid hormone, progesterone, signals the resumption of meiosis from the oocyte plasma membrane. In this review we re-examine the data on second messenger systems inXenopusoocytes and discuss some of the unresolved questions about hormone signal transduction during maturation. We outline some reasons for the contradictions in the literature and offer some suggestions for avenues of future research.
Differential accumulation of mRNA and interspersed RNA duringXenopusoogenesis and embrypgenesis
Authors: Liu Chengyu , Smith L. Dennis
Published: September 2008

Xenopusooctye cytoplasmic poly(A)+RNA has been shown to include two major complex classes: mRNA and interspersed RNA. the former is defined by its translatalility, the latter consists of non–translatable repeat–containing transcripts with unknown functions. In this study we compared the accumulation patterns of total mRNA and a subfamily of interspersed RNA, the XR family (McGrew&Richter, 1989, Dev. Biot. 134, 267–70)
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

To 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.
Transduction mechanisms for gonadotrophin-induced oocyte maturation in mammals
Authors: Mattioli Mauro
Published: September 2008

Although many validated techniques for oocyte maturation in vitro have been developed and eggs with normal development competence can be produced by these methods, the mechnism triggering oocyte maturation in vivo is still poorly understood. Little doubt exists that meiosis is reinitiated in pre-ovlulatory follicles by peak levels of gonadotrophins and that follicle somatic cells are directly involved since no receptors for gonadotrophins have been found on the oolemma.
Meiotic cycle checkpoints in mammalian oocytes
Authors: Fulka Josef , Bradshaw Judy , Moor Robert
Published: September 2008

Recent Spectacular achievements have enabled the identification of key molecules responsible for mitotic cell cycle progression through the stages of G1, the gap before DNA replication; S, the phase of DNA synthesis; G2, the gap before chromosome segregation; and M, mitosis itself. The last stage has been most intensively studied, where MPE, maturation promotion factor, has been found responsible for the nuclear events associated with chromosomal segregation and the prodcution of two identical daughter cells (see Murray & Hunt, 1993).
Sperm activation in species with external fertilisation
Authors: Tosit Elisabetta
Published: September 2008

The spermatoozoon is an excitable cell that responds to specific effectors by rapidly changing its behaviour. In species with external fertilisation, spermatozoa are stored in a quiescent state in the testis, but within seconds after spawning, dilution into water triggers several activation events such as increases in motility and respiration. In some species, these are followed by the acrosome reaction, an exocytotic process that allows the spermatozoon to penetrate the egg investments and activate the egg (Dale, 1983). The majority of information on sperm activation has come from the sea urchin; secondarily teleosts and starfish have proved to be useful models.
Fertility of mammalian spermatozoa: its development and relativity
Authors: Yanagimachi R.
Published: September 2008

Spermatozoa leaving the testis of normal animals are not ready to fertilise oocytes. They gain this ability while passing through the epididymis. It is interesting that spermatozoa of most animals take about 10 days to pass through the epididymis despite huge interspecies variations in the length of the epididymal tubule (e.g. some 30–50 m in large farm animals and about 1 m in the laboratory mouse) (Robaire & Hermo, 1988). The site where the spermatozoa begin to acquire fertilising capacity varies according to species, but it is generally the corpus epididymis or the proximal segment of the cauda epididymis where a large proportion of the spermatozoa become fertilisationcompetent. The distal segment of the cauda epididymis is the principal site for the storage of mature spermatozoa. Prolonged sojourn in the cauda epididymis (and vas deferens), however, could be detrimental to the spermatozoa.
Recent developments in cryopreservation of stallion semen with special emphasis on thawing procedure using thermal hysteresis proteins
Authors: Arav A. , Carney J.N. , Pease G.R. , Liu K.L.
Published: September 2008

While the first attempt to cool and preserve stallion semen was done 200 years ago (by Spallazani) and the first pregnancy achieved with frozen-thawes stallion semen almost 40 years ago (Barker & Gandier, 1957), frozen stallion semen has not been used widely, mainly because of its association with low fertility.
Embryonic stem cells in farm animals
Authors: Galli C. , Lazzari G. , Flechon J.E. , Moor R.M.
Published: September 2008

Embryonic stem cell technology is now well established in the mouse (reviewed by Robertson, 1987). This technology implies the isolation from the preimplantation embrao of a cell line (ES) that is cultured in vitro in an undifferentiated state. Embryonal carcinoma cells (EC) lines obtained from malignant tumours (Martin, 1975), together with all the information available on their culture requirements (reviewed by Heath, 1987), represented a very important starting point for the establishment of ES cells (Martin, 1981). ES cells share many characteristics with EC cells such as the ability to contribute to somatic tissues of animals obtained following injection of cells into a host blastocyst, to differentiate in vitro under appropriate stimuli (Rudnicki & McBurney, 1987) and to form retransplantable tumours. ES cells, however, have substantial advantages over EC cells in that they can be derived directly from a normal embryo, they maintain a normal karyotype and when reintroduced into a host blastocyst they can colonise the germ line (Bradley, 1987). ES cells are maintained in an undifferentiated state by the presence of feeder layers producing various factor(s) that prevent to the cells from differentiating. It has been shown that glycoproteins are responsible for this effect and these have been named according to their different activities: DIA, differentiation inhibitory activity (Smith & Hooper, 1987); LIF, leukaemia inhibiting factor (Smith et al, 1988; Williams et al, 1988); HILDA, human interleukin for DA cells (Moreau et al., 1988). It is now possible to establish and maintain ES cells in culture in the absence of feeders cells but in the presence of such factors (Nichols et al., 1990).
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

We 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

Polyclonal 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

The 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

Fresh 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

Sperm 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

Fusion 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

We 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

The 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

Under 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

The 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

Taurine 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

Cumulus-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

The 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

Fresh 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

It 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

Previous 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

We 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.
Structure and macromolecular composition of the zebrafish egg chorion
Authors: Bonsignorio Daniele , Perego Lucia , Giacco Luca Del , Cotelli Franco
Published: September 2008

The chorion is the acellular envelope surrounding mature eggs of teleostean fish. The macromolecular composition of the zebrafish (Danio rerio) egg chorion, organised as a three-layered structure, has been analysed. SDS-PAGE analysis, under reducing conditions, of isolated and purified chorions revealed a reproducible pattern of four major polypeptides (116, 97, 50 and 43kDa) and several minor bands. Lectin binding assays showed that both the 116 kDa and 50kDa proteins were recognised by concanavalin agglutinin (Con A), Galanthus nivalis agglutinin (GNA), Sambucus nigra bark agglutinin (SNA) and Ricinus communis agglutinin (RCA 120), suggesting that these polypeptides are N-linked glycoproteins. By contrast, neither the 97 kDa nor the 43 kDa polypeptides were stained by these lectins, indicating that these polypeptides are not glycosylated. Amino acid analysis also showed significant differences in the average content of some amino acids, for example serine and proline, when compared with previous reports.
Developmentally regulated markers ofin vitro-produced preimplantation bovine embroys
Authors: Shehu D. , Marsicano G. , Fléchon J.-E. , Galli C.
Published: September 2008

Expression of various developmentally regulated markers was screened throughout the preimplantation stages ofin vitro-derived bovine embryos. This was done by investigating the distribution of several nuclear, cytoplasmic and extracellular proteins by means of immunofluorescence microscopy. While lamin B appeared as a constitutive component of nuclei of all preimplantation stages, lamins A/C had a stage-related distribution. The early cleavage stage nuclei contained lamins A/C which generally disappeared in the following stages, with the possible exception of a few positive nuclei in the morula and early blastocyst stage. In the expanded blastocyst stage the nuclei of trophectoderm cells became positive while no positivity was observed in the inner cell mass cells. Starting from day 6, the appearance and/or polarised distribution of various cytoskeletal and cytoskeleton-related components such as Factin, α-catenin and E-cadherin gave an insight into the timing of events related to compaction of bovine e bryos. Compaction was correlated with the first differentiation event, i.e. the formation of trophectoderm; this is the first embryonic epithelium, characterised by cytokeratins and desmoplakin. Extracellular fibronectin was first detected in the early blastocyst stage shortly before the morphological differentiation of primitive endoderm, and in the later stages it was localised at the interface between trophectoderm and extraembryonic endoderm. Laminin and collagen IV were expressed by the endoderm cells and contributed to the extracellular matrix underlying the trophectoderm. This study is a first attempt to characterise the cells ofin vitro-derived bovine embryos valid for cell line derivation.
Assessment of the acrosome reastion inBufo arenarumSpermatozoa by immunostaining: comparison with other methods
Authors: Martínez María Laura , Cabada Marcelo O.
Published: September 2008

The acrosome reaction inBufo arenarumspermatozoa has been visualised only by electron microscopy. Different staining procedures for spermatozoa are described in the present study. By light microscopy the acorsomal status cannot be determined, In some reacted sperm, stained with Coomassie blue, a filament that could be the ‘perforatorium’ was observed, as seen by electron microscopy. Several fluorescent lectins were used, but only FITC-PNA binds to acrosomal proteins. However, the fluorescence observed was weak. Indirect immunofluorescence, with antibodies raised against acrosomal matrix, showed different staining patterns between acrosome-reacted and intact spermatozoa. The technique is specific for evaluating acrosomal status in a large population of spermatozoa. Moreover, immunostaining, in contrast with lectin staining, can be carried out in the presence of glycoconjugates such as oocyte extracellular matrix without interference.
Recombinant hamster sperm receptors that exhibit species-specific binding to sperm
Authors: Litscher Eveline S. , Wassarman Paul M.
Published: September 2008

Previous studies have shown that mouse sperm bind to hamster eggs and hamster sperm bind to mouse eggsin vitro. Furthermore, sperm receptor glycoprotein isolated from the zona pellucida of unfertilised hamster (hZP3) and mouse (mZP3) eggs binds to sperm from the heterologous species. Here, we expressed the hZP3 gene, under control of a constitutive promoter (pgk-1), in mouse embryonal carcinoma (EC) cells and Chinese hamster ovary (CHO) cells stably transfected with the hZP3 gene. In both cases, recombinant hZP3 (EC-hZP3 and CHO-hZP3) secreted into the culture medium was partially purified by high-performance liquid chromatography on a size-exclusion column and assayed for bioactivity using mouse and hamster gametes. Unlike hamster egg hZP3, which binds to both mouse and hamster sperm, EC-hZP3 and CHO-hZP3 exhibits species-specific binding to hamster sperm and induce hamster sperm, but not mouse sperm, to undergo the acrosome reactionin vitro. These results provide further evidence that species-specific binding of sperm to eggs in mammals is carbohydrate-mediated. Furthermore, the results suggest that recombinant forms of mammalian sperm receptors may be useful in assessing the molecular basis of species-specific fertilisation in mammals.
Tolerance of oocyte plasma membrane to electric current changes after fertilisation
Authors: Jing-he Tan , Zhong-hua Liu , Xing-shen Sun , Gui-xin He
Published: September 2008

The tolerance of the oocyte plasma membrane (oolemma) to electrical pulses (TEP) was investigated for oocytes, zygotes, and embryos at the early and late 2-cell stage. The oocyte survival rate after two electrical pulses (1.4KV/cm for 640μs each) was used as an indicator of the TEP of the oolemma. Survival rate of mid-pronuclear zygotes (94.3%±2.3%) was significantly (p< 0.05) higher than that of recently ovulated (2.1%±1.9%) andin vivoaged (25.1%±2.6%) oocytes; survival rate ofin vivoaged oocytes was also significantly higher than that of recently ovulated oocytes. Soon after fertilisation, the survival rate of the oocytes markedly increased, up to 94% at the mid-pronuclear stage. Survival ratedropped thereafter. These results suggest that the characteristics of the oocyte plasma membrane (oolemma) change after fertilisation.
Combined use of proacrosion immunocytochemistry and autosomal DNA in situ hybridisation for evaluvation of human ejaculated germ cells
Authors: Mendoza Carmen , Benkhalifa Moncef , Cohen-Bacrie Paul , Hazout André , Ménézo Yves , Tesarik Jan
Published: September 2008

The recently reported human pregnancies and births after fertilising oocytes with round spermatids recovered from the ejaculate of men with non-obstructive azoospermia have underscored the need for a more accurate evaluation of the nuclear and cytoplasmic maturation status of ejaculated germ cells. In this study we describe our first experience with a method combining the immunocytochemical visualisation of proacrosin with autosomal DNA fluorescencein situhybridisation (FISH) to assess ejaculated germ cells from patients with a spermiogenesis defect. The proacrosin immunoreactivity, analysed with the use of the monoclonal antibody 4D4, has been detected in cells of round spermatid size presenting a haploid FISH figure as well as in larger cells whose ploidy corresponds to primary and secondary spermatocytes. These observations are in agreement with previously published results obtained, with the use of the same antibody, by immunocytochemical analysis of histological sections of testicular tissue. All the cells of round spermatid size possessing proacrosin immunoreactivity were found to be haploid by FISH. On the other hand, some of the haploid cells of round spermatid size did not possess proacrosin immunoreactivity. The structural pattern of proacrosin immunoreactivity was highly variable both in spermatids and in younger spermatogenic cells. These data show that cell size is the main criterion to be used for the identification of ejaculated round spermatids, whereas the presence of the developing acrosome represents only an auxiliary criterion. The scoring of acrosomal development in ejaculated spermatids may be useful as part of pre-treatment diagnosis before the inclusion of infertile couples in a spermatid conception programme.
Oocyte-secreted factros regulate granulosa cell steroidogenesis
Authors: Vanderhyden Barbara C.
Published: September 2008

Investigations of strains of mice defective in germ cell development have revealed the importance of oocytes for the initial stages of folliculogenesis (Pellaset al., 1991; Huanget al., 1993). Various aspects of follicular development are dependent upon and/or influenced by the presence of oocytes, including granulosa cell proliferation (Vanderhydenet al., 1990, 1992) and cumulus expansion (Buccioneet al., 1990; Salustriet al., 1990; Vanderhydenet al., 1990; Vanderhyden, 1993). We are investigating the possibility that oocytes influence one of the primary functions of granulosa cells: steroidogenesis. In many species, granulosa cells removed from preovulatory follicles luteinisein vitro(Channinget al., 1982), presumably due to loss of contact with follicular luteinisation inhibitory factor(s). Indeed, follicular fluid can prevent granulosa cell luteinisationin vitro(Ledwitz-Rigbyet al., 1977). Follicular fluid, however, may simply be the medium for transport of factors secreted by oocytes to regulate granulosa cell activities.
Influence of vasoactive intestinal peptide (VIP), atrial natriuretic peptide (ANP) and insulin-like growth factor-I (IGF-I) on in vitro maturation of prepubertal and adult sheep oocytes
Authors: Ledda S. , Bogliolo L. , Leoni G. , Calvia P. , Naitana S.
Published: September 2008

Much effort has been focused on establishing optimal conditions for obtainingin vitromaturation of oocytes from different species with results comparable to those achieved afterin vivodevelopment (reviewed by Brackett, 1992). However, even though extraordinary progress has been made, thein vitrotechnology for oocyte maturation lags far behind thatin vivoand improvements are needed to increase the quantity and quality of the embryos produced from these matured oocytes.
Intracellular ion concentrations and their maintennance by Na+/K+-ATPase in preimplantation mouse embroys
Authors: Baltz Jay M. , Smith Stephen S. , Biggers John D. , Lechene Claude
Published: September 2008

We have measured the amounts of Na+, K+and C−in preimplantation mouse embryos (1-cell, 2-cell and morula) using electron probe X-ray microanalysis. The levels of these ions do not vary much over this period, and are approximately the same as those found in other mammalian cells, contrary to previous reports. We have confirmed that preimplantation embryos exhibit Na+/K+-ATPase activity at all stages examined, and have shown that the ATPase maintains high K+/Na+ratios (12–16) in all these embryonic stages, comparable to those seen in other healthy cells; this is in contrast to the low ratios reported in earlier work. Inhibition of the Na+/K+-ATPase results in the slow exchange of intracellular K+for extracellular Na+(half-time approximately 5 h), indicating that Na+/K+-ATPase activity maintains steep Na+and K+gradients in preimplantation mouse embryos as it does in most other cells.
Procaine-induced maturation ofXenopusoocytes is mediated by a transient activation of M-Phase promoting factor
Authors: Flament Stéphane , Bodart Jean-François , Browaeys Edith , Bertout Marc , Rousseau Arlette , Gannon Julian , Vilain Jean-Pierre
Published: September 2008

We have recently shown that the incubation ofXenopus laevisoocytes in procaine-containing solutions induced germinal vesicle breakdown without white spot formation and, in some cases, with the appearance of spindle and chromosomes in the cytoplasm. The present study was performed to determine whether M-phase promoting factor was involved in this unusual maturation. Procaine failed to induce maturation in the presence of 6-dimethylamino purine or roscovitine, which are both known to inhibit p34cdc2kinase. Histone H1 kinase activity was detected in procaine-treated oocytes but it was always lower than in progesterone-treated controls. A shift in p34cdc2was observed in oocytes that had been exposed to procaine for 16h, but it was not detected in those exposed for 24h. Finally, cytoplasm transfer experiments demonstrated that the maturation promoting activity that occurred in oocytes incubated in procaine for 16h could induce maturation of recipient stage VI oocytes. This transferable activity was weaker than that from progesterone-treated controls since only 30% of the recipients underwent germinal vesicle breakdown and only a few spindles were observed, which were not always correctly located. Taken together these results demonstrate that M-phase promoting factor is involved in the procaine maturing effect despite some differences compared with progesterone-treated oocytes which might explain the particular type of maturation induced by this substance. The discovery of the mechanisms by which procaine is able to activate M-phase promoting factor might now help in the understanding of some steps in progesterone-induced maturation that have still to be elucidated.
Virus-like particles, bacteria and microsporidia affect spindle-associated membranes in spermatocytes of Lepidoptera species
Authors: Wolf Klaus Werner
Published: September 2008

Larval testes of four Lepidoptera species were examined using electron microscopy. The testes of one species, the Mediterranean mealmothEphestia kuehniella(Pyralidae), were devoid of intracellular pathogens and serve as a control. In this species, metaphase spindles of primary spermatocytes showed a thick layer of perispindle membranes. The membranes were structurally very similar to the agranular endoplasmic reticulum. Membranes of this type occurred also at high frequency throughout the spindle matrix. The analysis of larval testes ofPieris brassicae(Pieridae) revealed virus-like particles within spermatocytes. In another species,Philudoria potatoria(Lasiocampidae), the spermatocytes possessed intracellular bacteria. Whereas the pathogens were found within the germ cells in these species, a fourth species,Plutella xylosteUa(Plutellide), showed microsporidia within somatic cells of the testis sheath. In all the infected animals, the mass of perispindle membranes was reduced in comparison with spermatocytes ofE. kuehniella. However, spindle structure appeared regular in the infected animals. This indicates that a thick layer of perispindle membranes is not decisive for spindle assembly and function in male meiosis of Lepidopera
Sperm nuclear envelope: breakdown of intrinsic envelope andde novoformation in hamster oocytes or eggs
Authors: Usui Noriko , Ogura Atsuo , Kimura Yasuyuki , Yanagimachi Ryuzo
Published: September 2008

During fertilisation of a fully mature oocyte, the sperm intrinsic nuclear envelope (SINE) disappears soon after sperm-oocyte fusion. A new nuclear envelope appears around the decondensed sperm chromatin when the oocyte reaches telophase II. Whether the SINE persists or rapidly disappears after sperm entery into immature oocytes or fertilised eggs has been controversial. Nuclear envelopes have been demonstrated around the sperm chromatin, which cannot be decondensed within the ooplasm of these oocytes or eggs, but whether these envelopes are persisting SINEs or newly formed envelopes has been apoint of dispute. To resolve this issue, the fate of the germinal vesicle stage(GV oocytes) or fertilised eggs at the pronuclear stage(PN eggs). The SINEs disappeared quikly within these oocytes or eggs, like those within maturing or mature oocytes, suggesting that the envelops around the sperm chromatin must be newly formed after SINE breakdown. To obtain further evidence, a detergent-treated, SINE-free sperm nucleus was injected into a PN egg. A new envelope appeared around the still-condensed or partially decondensed sperm chromatin within 3h after injection. Thus, disassembly of the SINE within ooplasm, unlike that of nuclear envelopes of other cells at prophase, is independent of the cell cycle stage of the oocyte or egg, whereas the ability of the ooplasm to assemble the new envelope is restricted to certain periods of the cycle. i.e. early prophase and telophase during meiosis and interphase, periods when active M-phase Promoting factor (MPF) is absent from the ooplasm.
Ultrastructural and immunocytochemical analysis of diploid germ cells isolated from fetal rabbit gonads
Authors: Moens André , Fléchon Bernadette , Degrouard Jéril , Vignon Xavier , Ding Jianchi , Fléchon Jacques-Edmond , Betteridge Keith James , Renard Jean-Paul
Published: September 2008

Germ cells were isolated from rabbit fetal gonads between 18 and 22 dayspost coitumand examined morphologically, ultrastructurally and for immunocytochemical and cytochemical characteristics. Observations were compared with the information available from the corresponding cells of other mammalian species. The general morphology and ultrastructure of healthy isolated rabbit fetal germ cells were found to be very similar to those of the rabbit and mouse diploid germ cellsin situ. Moreover, rabbit fetal germ cells shared common immunocytochemical characteristics with mouse undifferentiated embryonic stem cells or embryonic carcinoma cells, such as the presence of TEC-1 (SSEA-1) antigens, a peripheral network of F-actin, the absence of cytokeratins 8/18 and lamins A/C and an alkaline phosphatase activity. No difference between the sexes was observed. Morphological and physiological similarities with the migrating and cultured primordial germ cells of the mouse also suggest that diploid rabbit germ cells would be good candidates for deriving pluripotential embryonic germ cells (EG cells) if favourable culture conditions could be found. In conclusion, the rabbit may be suitable model for investigations on EG cells in domestic mammals with delayed meiosis.