bluuebunny/crossref_metadata_2025 huggingface dataset:
Long-lasting exocytosis and massive structural reorganisation in the egg periphery during cortical reaction inPlatynereis dumerilii(Annelida, Polychaeta)
Authors: Kluge B. , Lehmann-Greif M. , Fischer A.
Published: September 2008

The course of the cortical reaction in thePlatynereis dumeriliiegg is described from live observation and from sectioned fixed material and is found to differ in several aspects from the course of cortical reactions in better-known systems. Cortical granules are unusually numerous. They are discharged by exocytosis during a period of about 25 min following fertilisation (18°C). Most of the surplus membrane material brought to the egg surface by exocytosis is set free into the perivitelline space. Swelling of egg jelly precursor secreted by cortical granule exocytosis may be causal for the detachment of the vitelline envelope from the egg cell surface which, however, remains attached punctately to the vitelline envelope by about 30000 microvilli. Under the strain of the distending vitelline envelope, the bases of the microvilli move and line up, pulling the cell surface into a network of ridges. The grooves in between the ridges are the sites of exocytoses. Cytochalasin B, generally destabilising actin filaments, induces rupture of the microvilli and exaggerated distension of the vitelline envelope during the cortical reaction. In a final phase of the cortical reaction the vitelline envelope wrinkles and falls back onto the egg cell surface, the microvilli shorten and the egg cell transiently becomes deformed by local contractions. The cortical reaction in the nereid egg is discussed as a process of distortion and reorganisation of the egg cortex and plasmalemma. The abundance of cortical granules accommodating egg jelly precursor in thePlatynereisoocyte is attributed to the mode of so-called diffuse oogenesis characteristic of nereids, i.e. of differentiation of oocytes freely suspended in the coelomic fluid. In nereids, egg jelly therefore forms after fertilisation as opposed to ovulation.
The double spermatogenesis in scutigera coleptratrata (Myriapoda, Chilopoda): macro-and Microspermaltocyte growth
Authors: Pruescu Carol C. , Descamps Michel , Fabre Marie-Chantal , Serra Antoni
Published: September 2008

In Scutigera coleoptrata, double spermatogenesis (macro and micro germinal cells) was reinvesti
gted by means of both image analysis and electron microscopy. Image analysis of the cell-lines showed no differences in DNA content between the two types of spermatogesis.The only difference found, with the exception of the size and the related number of organelles, was that in the macro-cell line the chromatin was well dispersed and no condensed, which is the opposite of what was observed in the macro-cell line. It is asumed that the level of activity related to this condensation process is at the origin of the two pathways of development.
Control of the surface expression of uvomorulin after activation of mouse oocytes
Authors: Clayton Lesley , M.L. Josie , Martin McConnell , Johnson H.
Published: September 2008

Uvomorulin (E-cadherin) is the major cell adhesion molecule responsible for intercellular adhesion in early mouse embryos. In contrast to other cell adhesion molecules, it is not detectable on the cell surface until around 6 h after fertilisation or parthenogenetic activation, at the time when pronuclear formation occurs (Clayton, L., Stinchcombe, S.V. and Johnson, M.H., Zygote 1, 333–44, 1993). In order to investigate this developmental control of surface expression of uvomorulin, we examined the effects of inhibitors of various cellular processes on the appearance of uvomorulin at the oocyte surface, as assessed immunocytochemically. Inhibitors of cytoskeletal assembly (cytochalasin D and nocodazole), protein synthesis (puromycin and anisomycin), and DNA synthesis (aphidicolin) had no effect on surface expression. Brefeldin A, which inhibits intracellular transport and secretion, did prevent surface expression, but monensin did not. The effects of brefeldin were reversible; following 8 h of treatment, recovery of surface expression after removal of brefeldin began within 2 h. The time-course of surface expression post-activation suggested a link with pronuclear formation. However, when pronuclear formation was advanced experimentally using 6-dimethylaminopurine(DMAP), concomitant advancement of surface uvomorulin was not observed. Similarly, surface expression of uvomorulin did not accompany puromycin-induced pronuclear formation in maturing meiotic metaphase 1 (MI) oocytes in vitro. Thus, surface uvomorulin expression does not appear to be linked simply to pronuclear formation. Proteolytic processing of both newly synthesised and total uvomorulin to generate mature molecule from precursor increased within 30 min to 1 h after activation, and also occurred in the continued presence of brefeldin, suggesting that uvomorulin processing appears to be controlled independently of its suface expression.
Calcium influx, fertilisation potential and egg activation inFucus serratus
Authors: Roberts Stephen , Brownlee Colin
Published: September 2008

Fertilisation in the marine algaFucus serratusis accompanied by increased influx of Ca2+from the external medium. The onset of this increase, monitored with the Mn2+fluorescence quench technique, corresponded with the depolarisation phase of the fertilisation potential. External Ca2+was necessary for the onset of the fertilisation potential and the early activation events, including cell wall exocytosis. Removal of Ca2+from, or addition of Sr2+to, the external medium during the fertilisation potential reduced the magnitude of the depolarisation and prolonged its duration. While fertilisation potentials could not be elicited in the presence of 0.1 mM Ca2+, addition of Ba2+in the presence of 0.1 mM Ca2+allowed normal fertilisation potential and egg activation. Microinjection of ryanodine or cyclic guanosine 5'-monophosphate (cGMP) did not induce cytoplasmic Ca2+elevation or egg activation. Inositol 1,4,5-triphosphate [Ins(l,4,5)P3] produced a transient elevation of cytoplasmic Ca2+, monitored using ratio photometry, but did not cause cell wall exocytosis except at the site of microinjection. The results demonstrate an essential role for Ca2+influx duringFucusegg activation. The relative importance of influx and intracellular Ca2+release inFucusegg activation is discussed.
Localisation of two classes of acetylcholine receptor-like molecules in sperms of different animal species
Authors: Baccetti B. , Burrini A.G. , Collodel G. , Falugi C. , Moretti E. , Piomboni P.
Published: September 2008

The distribution of different classes of acetylcholine (ACh) receptor-like molecules in sperms of different invertebrate and vertebrate species is described. ACh receptor molecules belong to one of two classes: muscarinic receptors (mAChRs), associated with signal transduction mechanisms in the inner domain of the cell, and nicotinic receptors (nAChRs), capable of opening Na+channels when activated by the ligand. Molecules immunologically related to mAChRs and to ACh can be identified by specific antibodies, and revealed by immunofluorescent or immunogold staining; the nicotinic receptor-like molecules are localised as curare-sensitive affinity sites for α-bungarotoxin. In all species studied, both classes of receptors were found, with a similar distribution. Muscarinic-like molecules were found mainly in the sperm head regions of most species; such a localisation may be correlated to a function in sperm–egg interaction, for instance in the regulation of the block to polyspermy. Nicotinic-like molecules are present mainly in the tail and in the post-acrosomal region of most animals, thus confirming their function in the regulation of sperm propulsion, but are also present at the acrosomal region of most species. The distribution patterns of the different classes of molecules indicate that both may be in sperm–egg interactions, in addition to their known function in the regulation of sperm propulsion.
Different calcium-dependent pathways control fertilisation-triggered glycoside release and the cortical contraction in ascidian eggs
Authors: McDougall Alex , Sardet Christian , Lambert Charles. C
Published: September 2008

Fertilisation of ascidian eggs induces the rapid release of a cell surfaceN-acetylglycosaminidase that blocks sperm binding to vitelline coat sperm receptors resulting in a block to polyspermy. Fertilisation also triggers a large contraction of the egg (thus stimulating ooplasmic segregation) that is completed within 5 min of insemination. In eggs of the ascidianPhallusia mammillata, glycosidase release and cortical contractions are blocked by BAPTA-AM [bis-(o-aminophenoxy-ethane-N,N,N',N' -tetraacetic acid, tetra(acetoxymethyl)-ester], a cell-permeant calcium chelator, indicating that both processes are probaly dependent on a rise in intracellular calcium levels. Both glycosidase release and the cortical contraction are induced by treatment of the egg with the protein synthesis inhibitor emetine, while only the glycosidase release is induced by isoproterenol, carbachol or acetylcholine. Previous work with ryanodine demonstrated that ryanodine also caused glycosidase release but not the cortical contraction Inversely, activation by ionomycin in calcium-free sea water causes cortical contractions but not glycosidase release. Thus the two processes can be activated independently. Dextran-coupled (10kDa) calcium green-1 injected eggs show an increase in intracellular calcium 30–40s before the cortical contraction is triggered by fertilisation or ionomycin- induced activation. This confirms previous findings that the cortical contraction is a consequence of the activation calcium the triggered by te sperm. The extracellular calcium requirement for the glycosidase release suggests that calcium influx may be more important for this phase of egg activation. Thus activation eggs appears to involve two independent pathways involving calcium.
Pronuclear formation and intracellular glutathione content ofin vitro-matured porcine oocytes followingin vitrofertilisation and/or electrical activation
Authors: Funahashi Hiroaki , Stumpf Todd T. , Cantley Thomas C. , Kim Nam-Hyung , Day Billy N.
Published: September 2008

Pronuclear formation and intracellular content of glutathione, containing reduced and oxidised forms, in porcine oocytes maturedin vitrowere determined following insemination and/or electrical stimulation. After insemination, sperm penetration had occurred as early as 3 h and female pronuclei had formed by 6 h with complete development by 12 h. Male pronuclear formation occurred, primarily, between 9 and 12 h after insemination. Glutathione content of the oocytes decreased following sperm penetration and remained at a depressed level until 12 h. After electrical stimulation, oocyte activation had occurred and female pronuclei had formed by 3 and 6 h, respectively. Oocyte glutathione content did not change as a result of oocyte activation. When oocytes were exposed to an electrical pulse and then spermatozoa, female pronuclear formation was observed by 3 h after stimulation/insemination. Sperm penetration was observed between 3 and 9 h. However, the incidence of male pronuclear formation observed at 12 h was extremely low, although sperm decondensation had occurred in some oocytes. Oocyte glutathione content had not decreased by 6 h following electrical activation. These results demonstrate that the changes in glutathione content in porcine oocytes following fertilisationin vitrodiffer from those due to electrical activation. Further, the decreased intracellular glutathione content in oocytes activated by sperm penetration appears to be due to the presence of a sperm factor.
Intracytoplasmic sperm injection in dysmorphic human oocytes
Authors: Alikani Mina , Palermo Gianpiero , Adler Alexis , Bertoil Massimo , Blake Marlena , Cohen Jacques
Published: September 2008

Fertilisation and development of dysmorphic human oocytes recovered from hyperstimulated ovaries have been evaluated following intracytoplasmic sperm injection (ICSI) for treatment of male infertility. A total of 2968 oocytes at metaphase II of meiosis were injected, of which 806 (27.2%) were dysmorphic at the light microscopic level. Cytoplasmic abnormalities included granularity, areas of necrosis, organelle clustering, vacuoles, and accumulating saccules of smooth endoplasmic reticulum. Anomalies of the first polar body and zona pellucida, as well as non-spherical shapes of oocytes, were also noted. Contrary to previous findings linking some dysmorphisms to non-assisted fertilisation failure, in this study no single abnormality led to a reduction in the fertilisation rate, nor was fertilisation compromised in oocytes with multiple abnormalities. The incidence of normal fertilisation (two pronuclei and two polar bodies) was 69% in both the dysmorphic and non-dysmorphic oocytes. While overall pregnancy and implantation results were not altered in the group of patients (n = 242) in whom at least one dysmorphic oocyte was injected, exclusive replacement of embryos which originated from dysmorphic oocytes led to a higher incidence of early pregnancy loss. It is concluded that aberrations in the morphology of human oocytes – most probably a product of controlled ovarian stimulation – are of little or no consequence to fertilisation or early cleavage after ICSI. It is possible, however, that these embryos have a reduced potential for implantation and further development.
Comparison of the acrosome reaction-inducing ability of the outer and inner surfaces of the zona pellucida and oolemma: a study using the golden hamster
Authors: Yazawa Hiroyuki , Radaelli Giuseppe , Yanagimachi Ryuzo
Published: September 2008

The outer surface of hamster zona pellucida has a strong ability to induce the sperm acrosome reaction, whereas neither the inner surface of the zona nor the oolemma has this ability. Therefore, the spermatozoa that fertilise oocytes after subzonal insemination of spermatozoa (SUZI) must have been either acrosome-reacted prior to injection or acrosome-reacted spontaneously while swimming within the perivitelline space. The outer surface of fresh mouse zona is not capable of inducing the acrosome reaction of hamster spermatozoa. When fixed with glutaraldehyde, however, it became capable of inducing the hamster sperm acrosome reaction rather efficiently. The reason for this is not clear.
Estimation by radiation inactivation of the minimum functional size of acrosome-reaction-including substance (ARIS) in the starfish,Asterias amurensis
Authors: Ushiyama Akira , Chiba Kazuyoshi , Shima Akihiro , Hoshi Motonori
Published: September 2008

In the starfishAsterias amurensis, the jelly coat of the eggs contains a glycoprotein essential for the induction of the acrosome reaction in homologous spermatozoa that is termed the acrosome-reaction-inducing substance (ARIS).ARIS is a highly sulphated and fucose-rich glycoprotein of extremely high molecular mass(>104kDa). ARIS was irradiated with high-energy eletrons in order to estimate the minimum size required for its biological activity. The minimum functional unit or target size of ARIS was estimated to bec. 14 kDa by target size analysis. ARIS was significantly disintegrated by the irradiation, yet the total sugar content was not apparently reduced. The binding of125I-labelled ARIS to spermatozoa competed with that of irradiated ARIS, although the affinity of ARIS was much reduced after irradiation.
Calcium and cell cycle control in early embryos
Authors: Wilding Martin
Published: September 2008

Over the past few years, we have witnessed a burgeoning series of papers addressing the role of calcium signalling in cell cycle control. In this review I will attempt to bring together all the diverse threads and discuss new concepts that have arisen from the most recent data. Because the major part of the data concerns mitosis/meiosis entry and exit, I have focused on these areas. I will jointly refer to meiotic and mitotic phases of the cell cycle as M-phase because these phases are highly comparable. Studies of the cell cycle involve a huge range of species, from plants to humans. I will, however, restrict this review to the work performed in early embryos. I apologise in advance to contributors to this field whose names I do not mention because they do not work on embryos.
The effect of 6-dimethylaminopurine (6-DMAP) on DNA synthesis in activated mammalian oocytes
Authors: Ledda S. , Loi P. , Bogliolo L. , Moor R.M. , Fulka J.
Published: September 2008

Mouse, sheep and bovine metaphase II oocytes were briefly preincubated in medium with 6-dimethyl-aminopurine (6-DMAP) and thereafter activated with ethanol before further culture in 6-DMAP supplemented medium. The presence of 6-DMAP enhances the efficiency of activation and the speed of pronuclear formation but has no effect on DNA synthesis. These results are discussed in the context of recently published data showing that the preincubation of condensed chromosomes with 6-DMAP blocks DNA synthesis when nuclei become reformed.
Protein synthesis is not required for male pronuclear formation in bovine zygotes
Authors: Chian R.C. , Sirard M.A.
Published: September 2008

Following fertilisation, the sperm triggers a series of intracellular changes which initiate oocyte activation and pronuclear formation. Oocyte activation can also be induced artificially by several chemicals, such as the calcium ionophore A23187. The sperm nucleus is transformed into the male pronucleus through the interaction of oocyte cytoplasmic factors. The profile of protein synthesis is different in bovine oocytes following fertilisation and parthenogenetic activation. The formation of male and female pronuclei was not blocked by the presence of the protein synthesis inhibitor cycloheximide. These results suggest that bovine oocyte activation by sperm and parthenogenetic activation induce different cytoplasmic responses for protein synthesis and that new protein synthesis is not required for male pronuclear formation in bovine zygotes.
Bovine oocyte plasma membrane binding sites for sperm plasma membrane duringin vitrooocyte maturation and fertilisation
Authors: Wheeler Emily , Berger Trish , Behboodi Esmail
Published: September 2008

The experimental objective was to determine whether the capability of bovine oocyte plasma membrane to bind sperm changes duringin vitrooocyte maturation and fertilisation. Binding was quantified by the intensity of tetramethylrhodamine isothiocyanate (TRITC) fluorescence at the periphery of oocytes following incubation with biotinylated sperm plasma membrane proteins and subsequent incubation with TRITC-avidin. Bovine oocytes were maturedin vitro. Sample groups were removed after 0,6 and 22 h, or inseminated and further cultured for 24 or 48 h. Oocytes were denuded of cumulus cells and zona pellucida and co-incubated with 56 μg biotinylated bovine sperm plasma membrane protein for 45 min in 150 μl drops of saline-BSA. Controls were incubated for the same time period in the absence of sperm plasma membrane proteins. All oocytes were rinsed, incubated with TRITC-avidin and subsequently fixed and transferred to mounting medium. Oocytes were scanned with a confocal microscope and analysed using ImageQuant software. The binding of sperm plasma membrane was quantified by integrated fluorescent intensity in standardised ellipses spaced around the plasma membrane of the oocyte. Values are expressed as mean intensity units per 320 pixel ellipse. Binding of sperm plasma membrane continued to increase throughoutin vitrooocyte maturation and fertilisation (9051, 24318 and 49953 for 0 and 22 hin vitromatured oocytes and fertilised oocytes, respectively;p= 0.0001). A dramatic decrease in sperm plasma membrane binding to the oocyte plasma membrane was observed in 2-cell embryos (mean intensity = 24477,p= 0.0001). The observed binding was primarily due to the binding of sperm plasma membrane proteins, as control oocytes incubated with TRITC- avidin only were barely visible (integrated fluorescence intensity values ranged from 8 to 3757).
In vitrofertilisation of Chinese hamster oocytes by spermatozoa that have undergone ionophore A23187-induced acrosome reaction, and their subsequent development into blastocysts
Authors: Tateno Hiroyuki , Kamiguchi Yujiroh
Published: September 2008

To enhance potential use of the Chinese hamster,Cricetulus griseus, in developmental and cytogenetic studies of mammalian gametes and embryos, techniques forin vitrofertilisation and embryo culture were developed in the species. Spermatozoa were recovered from the vasa deferentia of mature males, and incubated in modified TYH medium for 1 h at 37°C under 5% CO2in air. They were then treated with ionophore A23187 (20¼M) for 10min to induce the acrosome reaction. Following ionophore treatment, superovulated oocytes were collected from hormonally stimulated females and incubated with the acrosome-reacted spermatozoa for 2 h at 37°C under 5% CO2in air. In this study, 245 oocytes ova (98.0%) were determined to be monospermic. The monospermic ova were then cultured in TYH supplemented with 1mM hypotaurine under the same gas phase. Within 30h of fertilisation, 182 ova (93.8%) cleaved to the 2-cell stage, and subsequently 163 ova (84.0%) developed beyond the 2-cell stage. Thus, obstinate developmental arrest at the 2-cell stage(‘2-cell block’) was not observed in this species. Ultimately, 65.5% of monospermic ova reached morula to blastocyst stages.
Cyclic AMP reversal of hypoxanthine-arrested preimplantation mouse embryos is EDTA-dependent
Authors: Dienhart Mary K. , Downs Stephen M.
Published: September 2008

Hypoxanthine can block preimplantation mouse embryo developmentin vitroat the 2- to 4-cell stages, and this has recently been shown to be reversed by cAMP-elevating agents. However, the extent of this hypoxanthine-induced arrest is determined by the culture conditions and strain of mouse. Whitten's and KSOM/AA are two embryo culture media that support preimplantation development to the blastocyst stage. This study was undertaken to examine the influence of several components in these media on hypoxanthine-arrested preimplantation mouse embryos and to test the hypothesis that reversal of the hypoxanthine block by cAMP-elevating agents requires cooperative interaction with the chelator, EDTA. Initial experiments demonstrated that embryo development was blocked in the presence of hypoxanthine in Whitten's medium but not in KSOM/AA; furthermore, removal of EDTA from KSOM/AA rendered this medium incapable of supporting high levels of development to blastocyst (9%), whereas high numbers of blastocysts (80%) formed in Whitten's medium, which does not contain the chelator. Consequently, Whitten's medium was used to test our hypothesis. It has previously been demonstrated that the phosphodiesterase inhibitor, IBMX, can reverse the developmental arrest imposed by hypoxanthine in EDTA-supplemented Earle's basic salt solution, but in the present study the addition of IBMX to Whitten's medium resulted in a block to development and failed to reverse the hypoxanthine arrest. These disparate effects can be explained by the presence or absence of EDTA. Supplementing Whitten's medium with EDTA reverses the IBMX effect, but not the hypoxanthine-induced block. While IBMX alone is unable to reverse the hypoxanthine block in Whitten's medium, development is greatly enhanced by the simultaneous addition of EDTA and IBMX. Similar results were obtained with the cAMP analogue, 8-AHA-cAMP. The data therefore support our hypothesis that the reversal of the hypoxanthine-induced arrest by cAMP-elevating agents is critically dependent on the presence of EDTA. We contrast this with the situation in mouse oocytes, where the hypoxanthine-induced meiotic arrest is not reversed by the addition of EDTA and/or cAMP-elevating agents.
DNA synthesis, microtubule and nuclear dynamics in porcine parthenotes
Authors: Liu Lin , Lee Caroline , Moor Robert M.
Published: September 2008

Parthenogenetically activated mammalian oocytes have been used in the past decade as cytoplasts, in an attempt to support the development of nuclear transplant embryos. The present experiments were undertaken to study the DNA synthesis and the organisation of microtubules, nuclear envelope and chromatin during the first cell cycle of electrically activated porcine oocytes (parthenotes) maturedin vitroby using immunocytochemistry and laser scanning confocal microscopy. The results showed that pronuclear-like (PN) formation began 4–5 h post-activation (hpa), whilst DNA synthesis as revealed by bromodeoxyuridine incorporation was initiated 5–6 hpa, with a maximum number of labelled oocytes (73%) around 11 hpa, and persisted in some parthenotes until 15–16 hpa. In the metaphase II (MII) oocytes, microtubules were detected only in the metaphase II spindle; no lamin A/C antigen was observed. Electrical DC pulses resulted in 91% of MII oocytes being activated and confocal microscopy Indicated that microtubules were assembled in the spindle first for the extrusion of a second polar body, and for the second time for division from one to two cells. Nuclear envelope, indicated by anti-lamin A/C stain, was formed around the time of PN formation and surrounded the nuclear chromatin of 1- and 2-cell parthenogenotea. These results demonstrate that the apparent normality in both DNA synthesis and dynamics of microtubules and nuclear envelope is involved with chromosomal organisation in the parthenotes. In addition, the use of electrically activated IVM oocytes for both nuclear transfer and parthenogenetic studies in pigs is discussed.
Meiosis reinitiation in Ruditapes philippinarum (Mollusca): involvement of L-calcium channels in the release of metaphase I block
Authors: Moreau Marc , Leclerc Catherine , Guerrier Pierre
Published: September 2008

Prophase-arrested oocytes of Ruditapes philippinarum cannot be fertilised or stimulated by excess KCl, in contrast to the situation found in other bivalve species such as Barnea and Spisula. However, these oocytes can be triggered to reinitiate meiosis following treatment by serotonin or several pharmacological agents (calcium ionophores, thapsigargin, weak bases) which promote an intracellular calcium surge. Ruditapes oocytes further arrest in metaphase I, at which stage they can be activated either by sperm or by excess KCl. This suggests that functional voltage-operated calcium channels are expressed in this species during the course of maturation. Using pharmacological tools and direct binding of specific dihydropyridines, we demonstrate that these channels are L-type calcium channels which become functional after serotonin stimulation, their number increasing before germinal vesicle breakdown. Moreover we establish that: (1) the addition of 20 μM S(—)Bayκ8644, an agonist of L-type calcium channels, to metaphase-arrested oocytes releases them from metaphase block; (2) incubating these oocytes with PN200-110, a potent blocker of L-type calcium channels, inhibits their activation by both excess KCl and fertilisation. Taken together these data suggest that the absence of L-type calcium channels in the membrane of prophase-arrested oocytes of Ruditapes may account for their inability to be fertilised.
Differential distributions of two adducin-like protein isoforms in theDrosophilaovary and early embryo
Authors: Zaccai Michèle , Lipshitz Howard D.
Published: September 2008

Adducin is a cytoskeletal protein that can functionin vitroto bundle F-actin and to control the assembly of the F-actin/spectrin cytoskeletal network. TheDrosophila Adducin-like(Add) locus (also referred to ashu-li tai shao(hts)) encodes a family of proteins of which several are homologous to mammalian adducin (Dinget al., Proc. Natl. Acad. Sci. USA90, 2512–16, 1993; Yue & Spradling,Genes Dev.6, 2443–54, 1992). We report the identification of two novel adducin isoforms: a 95 × 103Mrform (ADD-95) and an 87 × 103Mrform (ADD-87). We present a detailed analysis of the distribution patterns of ADD-95 and ADD-87 during oogenesis and embryogenesis. The isoforms are co-expressed in several cell- and tissuetypes; however, only ADD-87 is present in mid- to late-stage oocytes. ADD-87 is present throughout the oocyte cortex at stages 9 and 10 of oogenesis but is detectable only at the anterior pole from stage 11 onward, correlated with localisation ofAdd-htsmRNA first to the cortex and then to the anterior pole of the oocyte. ADD-87 co-localises with F-actin and spectrin in the cortex of the oocyte through stage 10 of oogenesis, consistent with a possible role in cytoskeletal assembly or function predicted by mammalian studies.
Penetration of concanavalin-A-treated Chinese hamster oocytes by golden hamster spermatozoa in vitro, and chromosome analysis of hybrid 1-cell zygotes
Authors: Watanabe Seiji , Tateno Hiroyuki , Kamiguchi Yujiroh
Published: September 2008

Pretreatment of zona-free Chinese hamster (CH) oocytes with three kinds of lectin – concanavalin A (Con-A), phytohaemagglutinin-P (PHA) and wheat germ agglutinin (WGA) – was attempted in order to improve penetration by golden hamster (GH) spermatozoa in vitro. Con-A had no significant effect on penetration at 2 μg/ml, adequately facilitated oocyte-sperm fusion at 4 μg/ml, and caused excessive sperm binding and resultant severe polyspermy at 10 μg/ml. Neither PHA nor WGA had positive effects on sperm penetration at any concentrations (2–10 μg/ml) examined. Using the Con-A (4 μg/ml)pretreatment, high rates of interspecific fertilisation and subsequent chromosome analysis of hybrid 1-cell zygotes were achieved. Among 258 CH oocytes used, 212 (82.2%) were fertilised and 153 (72.2% of fertilised ova) developed to the first cleavage metaphase. Eventually, 132 CH-derived chromosome complements and 153 GH-derived ones were successfully karyoanalysed. Incidences of aneuploidy and structural anomaly were 3.1% and 2.3% in CH complements, and 1.4% and 6.5% in GH complements, respectively. These incidences were not significantly different from those obtained by intraspecific in vivo fertilisation, suggesting that our interspecific in vitro fertilisation system does not cause chromosome aberrations.
Mitogen-activated protein kinase activity and microtuble organisation are altered by protein synthesis inhibition in maturing porcine oocytes
Authors: Inoue Maki , Naito Kunihiko , Nakayama Taisuke , Sato Eimei
Published: September 2008

Previously we have shown that mitogen-activated protein (MAP) kinase activity abruptly increases at the first metaphase (M1) and remains significantly higher than that at the germinal vesicle (GV) stage until the second metaphase (M2) in porcine oocytes culturedin vitro. The present paper describes how the mechanism of the blockage of meiotic maturation by protein sythesis inhibition involves MAP kinase regulation. Cycloheximide arrested both germinal vesicle breakdown (GVBD) and the normal transition from M1 to M2. MAP kinase activation was also reduced in these maturation-inhibited oocytes. By using immunofluorescence microscopy with the monoclonal antibody raised against rat α-tubulin, we showed that cycloheximide caused morphological abnormality in a spindle at M1, but not at M2. All these results indicate that in porcine oocytes: (1) GV blockage by protein synthesis inhibition involves the suppression of both histone H1 kinase and MAP kinase activation, (2) during the transition from M1 to M2, maintenance of a normal metaphasic spindle and high MAP kinase activity require protein synthesis, and (3) once the M2 cytoskeletal structures have been completed, and/or after the ‘critical period’, cytostatic factor activity is independent of protein synthesis.
Maturation of hamster oocytes under chemically defined conditions and sperm penetration through the zona pellucida
Authors: Kito Seiji , Bavister Barry
Published: September 2008

This study aimed to achieve high frequencies of nuclear maturation and penetrability through the zona pellucida of hamster oocytes cultured under protein-free conditions. Completion of nuclear maturation by cumulus-intact, immature oocytes (79% metaphase II stage) was depressed (37%p< 0·05) by adding four amino acids (glutamine, isoleucine, methionine and phenylalanine) reported necessary for nuclear maturation of cumulus-free oocytes. Followingin vitromaturation, cumulus cells were removed and oocytes were inseminated with capacitated sperm, but after 6 h sperm:egg co-incubation, only 24% ofin vitromatured oocytes were penetrated compared with 60% ofin vivomatured oocytes (p< 0·05). Time required for zona lysis by α-chymotrypsin was not significantly different amongin vitroandin vivomatured oocytes and 1-cell embryos. Addition to the maturation medium of soybean trypsin inhibitor or fetuin, both known to inhibit the zona reactionin vitro, did not improve penetrability ofin vitromatured oocytes, implying that in hamsters, unlike other rodent species, a premature zona reaction is unlikely to be responsible for inhibiting sperm penetration. When oocytes were incubated with 20% periovulatory oviductal fluid (OF) for another 3 h after maturation, penetration was significantly improved (60% vs 37% with and without OF, respectively;p< 0·05), but was not equivalent to penetration ofin vivomatured follicular oocytes similarly treated with OF (84%, p < 0·05)However, zona penetration was further improved by increasing sperm concentration from 1·0 × 104(66%) to 5·0 or 10·0 × 104sperm/ml (89%, p < 0·05). This study shows that nuclear maturation of hamster oocytes can occur in chemically defined medium, and indicates that a deficiency in the zona ofin vitromatured oocytes can be overcome by preincubation with OF and insemination at high sperm conccentration.
Expression of c-fosand c-junproto-oncogenes by ovine preimplantation embryos
Authors: Wu Bin
Published: September 2008

The c-fosand c-junproto-oncogenes are involved in the regulation of gene expression, cell proliferation, differentiation and tumorigenesis. Embryogenesis, like tumorigenesis, involves dramatic cell growth, cleavage and differentiation processes. Activation of the c-fosand c-junproto-oncogenes in sheep conceptuses during the period of rapid growth and elongation was examined using reverse transcription and polymerase chain reaction (RT-PCR). The specificity of PCR products was determined by Southern blot hybridisation analysis with a non-radioactive DNA probe. A band corresponding to a 507 bp fragment (predicted amplified c-fosgene cDNA product) was observed in 3 of 9 day-13, 1 of 4 day-14 and 1 of 2 day-16 embryos. Meanwhile, a 400 bp c-juntranscript was aslo detected in 1 or 2 day-12, 3 of 9 day-13 and 2 of 2 day-16 embroyos. These results suggest that mRNA transcripts of c-fosand c-junproto-oncogenes were specifically expressed during the period of ovine embryonic elongation and may have a possible role in preimplantation embryonic development of sheep.
RNase A activity in Japanese quail oocytes
Authors: Stepińska Urszula , Malewska Arleta , Olszańska Bożenna
Published: September 2008

The presence of RNase A activity was studiedin vitroin homogenates of quail oocytes and early embryos using [3H]poly(U) as a substrate. The activity was measured by adsorption of the undegraded substrate onto DE-81 filter paper discs and by chromatographic separation on a Sephadex G-50 column. RNase A activity examined by these methods was almost undetectable in quail previtellogenic, vitellogenic and ovulated oocytes as well as in the embryos from laid eggs. It is estimated to be about 1.1 × 10−5Kunitz units per ovulated oocyte. Higher activity starts to appear in gastrulating embryos. These findings are discussed in relation to other studies demonstrating the high stability of maternal RNA during early development, especially in growing oocytes.
Co-culture with pig membrana granulosa cells modulates the activity of cdc2 and MAP kinase in maturing cattle oocytes
Authors: Motlík Jan , Šutovský Peter , Kalous Jaroslav , Kubelka Michal , Moos Jiří , Schultz Richard M.
Published: September 2008

Bovine cumulus-enclosed oocytes, initially cultured up to diakinesis (8h of initial culture) or metaphase I (12h of initial culture), were subsequently co-cultured for 6 h in contact with pig membrana granulosa (PMG) cells and then assayed for histone H1 and MAP kinase activities. In addition, the phosphorylation state of ERK 1,2 proteins was determined by Western blotting. The alterations in nuclear envelope breakdown, meiotic spindle formation and the patterns of chromosome condensation were analysed by immunofluorescence and transmission electron microscopy. The diakinesis-stage oocytes (initially cultured for 8h) already possessed high histone H1 kinase and MAP kinase activities that were correlated with condensed and partially individualised chromosomes. The ERK 1 and most ERK 2 proteins were partly phosphorylated. Following the 6h co-culture of these oocytes with PMG a rapid decrease in MAP kinase activity and a slower decrease in histone H1 kinase occurred, as well as ERK 1 and ERK 2 dephosphorylation. Both kinase activities and ERK 1,2 phosphorylation were fully restored following the release of the oocytes from co-culture and a subsequent culture in the absence of PMG. Moreover, the clumped bivalents were reindividualised and 56% of these oocytes reached metaphase II after 20 h of culture without PMG. The metaphase I oocytes, initially cultured for 12 h, displayed a fusiform meiotic spindle and a metaphase array of chromosomal bivalents, accompanied by high levels of both histone H1 and MAP kinase activity. Co-culture of MI oocytes with PMG abolished the activity of both kinases and caused the dephosphorylation of ERK 1 and ERK 2. Furthermore, the spindle microtubules were depolymerised and the chromosomal bivalents clumped into a single mass. Neither of the protein kinase activities nor the meiotic spindle were restored following subsequent culture in the absence of PMG for up to 20 h. These observations indicate that underin vitroconditions membrana granulosa cells can cause a prompt decrease in histone H1 and MAP kinase activities, and metaphase I oocytes. While these events are fully reversible in late diakinesis oocytes, metaphase I oocytes did not complete maturation after release from co-culture.
Spontaneous maturation in Bufo arenarum oocytes: participation of protein kinase C
Authors: Zelarayán L. , Oterino J. , Bühler M.I.
Published: September 2008

Although progesterone is the maturation inducer in amphibians, it has been demonstrated that inBufo arenarumoocytes resumed meiosis with no need of exogenous hormonal stimulus if derived of their enveloping, follicle cells. This phenomenon, called spontaneous maturation, is quite rare in amphibians. InB. arenarum, spontaneous maturation took place only in oocytes obtained during the reproductive period (spring-summer). During this period the oocytes also demonstrated a respiratory activity characteristic of mature oocytes. Interestingly, full-grownB. arenarumoocytes always responded to progesterone regardless of the season in which they were obtained and of their respiratory activity. The disposition of oocytes competent or not competent to mature spontaneously provides a useful system for the study of molecular mechanisms involved in the maturation process. The data presented here indicate that the activation of protein kinase C (PKC) induces germinal vesicle breakdown (GVBD) in denuded oocytes unable to mature spontaneously (winter oocytes) and is involved in the in vitro spontaneous maturation ofB. arenarumfull-grown oocytes. The inhibition of PKC by 1-(5-isoquinolynyl-sulphonyl-2-methyl-piperazine (H-7) impeded spontaneous maturation in a dose-dependent manner, thus supporting the participation of the PKC pathway during this process. Interestingly phorbol 12-myristate-13-acetate (PMA)-induced GVBD is inhibited by the incubation of the oocytes in dibutyryl cAMP (dbcAMP), indicating that both pathways, PKC and protein kinase A (PKA), are related at a certain point. However, spontaneous GVBD is less sensitive than PMA-induced GVBD to dbcAMP. This fact would support the suggestion that in spontaneous GVBD mechanisms different from activation of PKC are at work.
Unprotected freezing of human spermatozoa exerts a detrimental effect on their oocyte activating capacity and chromosome integrity
Authors: Rybouchkin Andrei V. , De Sutter Paul , Dhont Marc
Published: September 2008

The influence of unprotected freezing of mammalian spermatozoa on their oocyte activating capacity and chromosome integrity is unknown. However, this type of sperm treatment has been used in assisted reproduction by intracytoplasmic sperm injection in cattle and humans. The mouse oocyte injection test was used to analyse the influence of unprotected freezing of human spermatozoa on their reproductive characteristics. Mouse oocytes were microinjected with intact human spermatozoa or spermatozoa treated with two cycles of unprotected freeze-thawing. Oocytes surviving the injection were either cultured without further treatment or exposed to ethanol solution to induce parthenogenetic activation. Both injected and activated oocytes were used for sperm chromosome analysis. The results revealed a significant reduction in oocyte activating capacity and a tenfold increase in the incidence of structural chromosomal abnormalities in human spermatozoa treated by unprotected freezing. We conclude that unprotected freezing of human spermatozoa has a detrimental effect on their reproductive characteristics. Our data also provide a new perspective on the stability of mammalian spermatozoa to physical factors and demonstrate the importance of detailed analysis of the stability of sperm structures for successful development of new approaches in assisted reproduction.
Antral follicles confer developmental competence on oocytes
Authors: Moor R.M. , Lee C , Dai Y.F. , Fulka J
Published: September 2008

This paper addresses the proposition, first advanced by Wilson (1925), that successful embryogenesis depends on an ordered series of events in oogenesis. It is at the completion of this varied set of intracellular changes that the oocyte finally acquires its full capacity to support fertilisation and development. Amongst the earliest nuclear events are those associated with chromosome pairing and meiotic recombination. During the growth phase cell volume increases 300-fold and the cytoplasm becomes the storage site for RNA and protein which will be mobilised during early development. Finally, a short phase of intracellular reprogramming, or maturation, completes the series of events during oogenesis that confer developmental competence upon the oocyte. Follicle cell support is an indispensable requirement for ordered oocyte development and provides the early germline cell with many of the essential nutrients and growth regulators required to ensure progression through the protracted growth phase (see contributions by Cecconi & Rosella and De Feliciet al.this issue). Although different, the interactions between the full-grown oocyte and the antral follicle are no less crucial to the acquisition of competence than those involved in the earlier stages of oogenesis.
Control of ovarian innervation
Authors: Gioia Luisa
Published: September 2008

The presence of intra-ovarian nerves was reported more than a century ago and successive investigations have demonstrated that the mammalian ovary is innervated by both sympathetic and sensory fibres distributed to the different compartments of the gland, such as blood vessels, ovarian stroma and follicle wall. Despite the extensive ovarian innervation and the experimental evidence indicating that neuromediators can influence sex steroid production, the role of the nervous system in the control of ovarian activity is still largely unknown.
Influence of granulosa cells and of different somatic cell types on mammalian oocyte development in vitro
Authors: Cecconi Sadra , Colonna Rosella
Published: September 2008

In mammals the ability of an oocyte to become fertilised is the result of a complex process occurring within the ovarian follicle which depends on the stagespecific expression of oocyte genes and the presence of granulosa cells (for a review see Buccioneet al., 1990a). The coordinated development of germinal and somatic components of the follicle is regulated by two principal systems of interaction, based on the presence of gap junctions and on the production of paracrine factors. Gap junctions link granulosa cells to each other and to the oocyte (Anderson & Albertini, 1976), and represent a major route for the transfer of small molecules involved in oocyte metabolism (for a review see Mangiaet al., 1992) and regulation of the arrest and resumption of meiosis (for a review see Eppig, 1993). The production of paracrine factors by granulosa cells has been suggested by the findings that these cells express the production of theSteellocus, the Steel factor (SLF) or kit ligand (KL; Motroet al., 1991; Manovaet al., 1993), and that this factor promotes oocyte growthin vitrowhen used at high concentrations (Packeret al., 1994). Since KL is too large to be transmitted through gap junctions, it must necessarily be released in the extracellular environment before binding to the c-kitreceptor present on oocyte membrane (Manovaet al., 1990; Horieet al., 1991).
Participation of nerve growth factor in the regulation of ovarian function
Authors: Dissen Gregory A , Mayerhofer Artur , Ojeda Sergio R.
Published: September 2008

Nerve growth factor (NGF) belongs to a family of related target-derived proteins required for the survival and development of discrete neuronal populations in the central and peripheral nervous systems (Levi-Montalcini, 1987; Snider, 1994). Although initial observations led to the conclusion that the biological actions of neurotrophins are restricted to the nervous system (Thoenen, 1991; Raffioniet al.1993), new evidence suggests that they, and in particular NGF, can also affect non-neuronal cells.
Endocrine and paracrine regulation of cumulus expansion
Authors: Salustri Antonietta , Camaioni Antonella , D'Alessandris Cristina
Published: September 2008

In a Graafian follicle, granulosa cells are classified into two principal cell subpopulations: cumulus cells, which are closely associated with the oocyte to form the cumulus cell-oocyte complex (COC), and mural granulosa cells, which are organised as a stratified epithelium at the periphery of the follicle. Following the preovulatory gonadotropin surge, cumulus cells lose contact with mural granulosa cells and start to synthesise and secrete a large amount of hyaluronan (HA), a glycosaminoglycan with high molecular weight and large hydrodynamic domains (Salustriet al., 1992). Proteins derived from serum (Chenet al., 1992, 1994) and synthesised by cumulus cells (Camaioniet al., 1993, 1996) organise the strands of HA into an intercellular elastic network that traps the cumulus cells and the oocyte in a unit which can not be mechanically dissociated – a process also referred to as cumulus expansion. At ovulation, the expanded COC is released through the ruptured follicle wall and transferred to the oviduct. The matrix in the expanded COC facilitates its extrusion from the follicle and its capture by oviductal fimbria, and provides, together with the cumulus cells, a suitable microenvironment for sperm penetration and fertilisation (for references see Salustriet al., 1993).
Intra-ovarian regulation oocyte developmental competence in cattle
Authors: Gandolfi Fulvio
Published: September 2008

Each oestrous cycle the ovary produces one or more oocytes, depending on the species considered, which are fully competent to sustain the development of a new individual and are defined as matured. Ovulation is the terminal phase of a lengthy and complex selection process which enables only a minute proportion of the oocytes present in the ovary to be ovulated and possibly fertilised. The use of exogenous gonadotrophins for the pharmacological stimulation of the ovaries has clearly indicated that oocytes naturally excluded by the selection process can also be matured and, if fertilisedin vivoorin vitro, can generate normal offspring.