bluuebunny/crossref_metadata_2025_split huggingface dataset:
Structure and macromolecular composition of the zebrafish egg chorion
Authors: Bonsignorio Daniele , Perego Lucia , Giacco Luca Del , Cotelli Franco 
Published: September 2008

SummaryThe 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

SummaryExpression 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

SummaryThe 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

SummaryPrevious 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

SummaryThe 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&lt; 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

SummaryThe 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.
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

SummaryWe 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

SummaryWe 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

SummaryLarval 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

SummaryDuring 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

SummaryGerm 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.
Effects of oviductal fluid and heparin on fertility and characteristics of porcine spermatozoa
Authors: Kim Nam-Hyung , Day Billy N. , Lim Joon-Gyo , Lee Hoon Taek , Chung Kil Saeng 
Published: September 2008

SummaryThe objective of this study was to determine the effects of oviductal fluid and heparin on sperm penetration and the characteristics of spermatozoa. The addition of oviductal fluid and heparin to the fertilisation medium decreased sperm penetration and the mean number of spermatozoa in penetrated eggs. The number of spermatozoa firmly bound to zona pellucida was also decreased in the presence of oviductal fluid and heparin. Chlortetracycline (CTC) fluorescence patterns were used to determine the incidence of capacitation and the acrosome reaction. The proportion of capacitated and acrosome-free spermatozoa increased when spermatozoa were exposed for 1.5 and 3 h to oviductal fluid and heparin. In contrast heparin alone did not increase the number of capacitated spermatozoa at these time points. These results suggest that factor(s) in oviductal secretions reduce polyspermic fertilisation and the number of spermatozoa that will penetrate porcine oocytes. The reduction of polyspermic penetration by oviductal secretions may be due to a reduced number of spermatozoa in the fertilisation medium with an intact acrosome.
Intermediate filament proteins immunologically related to cytokeratins in the oocyte of the fishCyprinus carpio
Authors: Mencarelli Caterina , Cotelli Franco 
Published: September 2008

SummaryWe have used monoclonal antibodies specific for different sets of human cytokeratins and the anti-IFA (Intermediate Filament Antigen) antibody to investigate the expression of intermediate filament proteins in the mature oocyte of the teleostCyprinus carpio. Several polypeptides have been identified, showing molecular weights ranging from 43 to 65kDa. Two-dimensional analysis of the immunoreactive species revealed the presence of at least six major protein spots and a series of minor components, grouped in quite a narrow pI range from 5.52 to 6.28. The general complexity of the carp oocyte cytokeratin-related cytoskeleton appears to be higher than those described for oocytes of other vertebrate species.
The behaviour of mitotic nuclei after transplantation to early meiotic ooplasts or mitotic cytoplasts
Authors: Kubelka Michal , Moor Robert M. 
Published: September 2008

SummaryThis study evaluates the ability of the cytoplasm to determine the nature of the division cycle (meiotic or mitotic) in nuclei obtained from mitotically dividing cells. Using mouse oocytes in different stages of development two types of cytoplasm were prepared: firstly, early meiotic ooplasts were obtained by enucleation of non-matured, prophase-stage oocytes; secondly, mitotic cytoplasts were prepared by enucleation and activation of metaphase II (Mll)-stage oocytes. These two types of cytoplasts were then used in fusion experiments, in which mouse primitive type A spermatogonia (prospermatogonia) or mouse fibroblasts were used as a source of donor nuclei. While the fusion of prospermatogonia with mitotic cytoplasts resulted, as expected, in normal premature chromosome condensation (PCC) and subsequent pronuclear formation (58.1%), the majority of hybrids obtained by fusion of prospermatogonia with early meiotic ooplasts (40.3%) displayed unique morphology consisting of two sets of chromosomes organised in two spindle centres connected by microtubules. Each set of chromosomes contained the haploid (1n) number of chromosomes as revealed by chromosome analyses. The same morphology was observed also in 44.2% of hybrids in which the differentiated nuclei of fibroblasts were used as a source of donor mitotic nuclei. In both cases the hybrids were blocked at this stage with high activity of maturation promoting factor (MPF), resistant to any kind of activation and not able to undergo further development. These results suggest that the early meiotic ooplasm was able to induce the initiation of a meiosis-like reducing division in mitotic nuclei originating both from the germline cells and from more differentiated somatic cells.
The response of mouse oocytes injected with sea urchin spermatozoa
Authors: Wakayama T. , Uehara T. , Hayashi Y. , Yanagimachi R. 
Published: September 2008

SummarySea urchin spermatozoa were injected into mature mouse oocytes to determine whether they can activate mouse oocytes and, if so, how they behave within the oocyte cytoplasm of such a distant species. While injection of a single spermatozoon into each oocyte did not activate any of the oocytes, injection of 10 spermatozoa activated about 20%. Within the cytoplasm of unactivated oocytes, sperm heads commonly transformed into chromosome-like structures. When a single spermatozoon was injected, and oocytes were then activated by Sr2+, about 30% of the activated oocytes had both female (mouse) and male (sea urchin) pronuclei when examined 8 h after sperm injection. These results indicated that sperm-borne oocyte activating factor(s) and the cytoplasmic factors controlling the development of the sperm pronucleus are not strictly species-specific.
Sperm head decondensation, pronuclear formation, cleavage and embryonic development following intracytoplasmic injection of mitochondria-damaged sperm in mammals
Authors: Ahmadi Ali , Ng Soon-Chye 
Published: September 2008

SummaryThe objective of this study was to investigate the influence of sperm mitochondrial destruction on sperm head decondensation, male pronuclear formation, cleavage and embryonic development. In the study two models were used:heterologous (hamster ICSI assay: human sperm injected into a hamster oocyte) for evaluation of sperm head decondensation and pronuclear formation, and homologous (mouse model) for the study of fertilisation and development. Destruction of mitochondria of the sperm was achieved by exposure to cyanide, a respiratory poison. Rhodamine 123 was used to evaluate the functional integrity of mitochondria. Sperm head decondensation was found to be not statistically significantly affected by mitochondrial damage (p= 0.8), with 62.8% and 67.9% condensa tion in the experimental and control groups respectively. Male pronucleus formation was seen in 40.2% and 44.4% of the injected oocytes in the experimental and control groups respectively. In the mouse experiments 45.5% and 49.7% of the injected oocytes were fertilised in the mitochondria-damaged and live-intact sperm groups respectively(p = 0.53)Development to blastocyst was achieved in 53.5% and 59.4% of the experimental and control groups respectively; the difference was not significant (p= 0.71). Inner cell mass (ICM) cell number were 15.7 ± 4.02 and 43.1 ± 11.3 respectively in the mitochondria damaged group; the equivalent numbers were 14.12±4.12 and 39.3 ± 12.6 in the control group. However, the differences in ICM and total cell counts between these two groups were not significant. Of the blastocysts transferred to pseudopregnant mice, 51.3% (20/36) implanted and 33.4% (12/36) developed to live fetuses in the mitochondria-damaged group. These rates were 60.5% (23/38) and 39.5% (15/38) in the control group. In conclusion, this study shows that functional integrity of thesperm mitochondria is not necessary in the process of fertilisation and development when the sperm is deposited into the ooplasm. Fertilisation and development can be achieved by injection of sperm at the very early stage of necrosis in which only the mitochondria have been destroyed and the rest of the cell including the plasma membrane is still intact.