bluuebunny/crossref_metadata_2025_split huggingface dataset:
Characterization of the antiviral activity constitutively produced by murine conceptuses: Absence of placental mrnas for interferon alpha and beta
Authors: Cross J. C. , Farin C. E. , Sharif S. F. , Roberts R. M. 
Published: February 2005

AbstractAntiviral activity has been found in conceptus and placental tissues in numerous species, including mice, pigs, sheep, cattle and humans. In sheep and cattle, the antiviral activity is due to an interferon alpha (IFN‐α), but in other species the nature of the protein(s) responsible for placental activity is unknown. The objectives of this study were to determine if the constitutive antiviral activity associated with the mouse conceptus is produced as early as the peri‐implantation period, and to determine if the activity is due to an IFN‐α or ‐β. Conceptus and placental tissue explants released antiviral activity from Day 4 through at least Day 16 of gestation as measured in an agar overlay bioassay employing CHO cells challenged with vesicular stomatitis virus. This activity was neutralized by antiserum against MuIFN‐α/β. The same antiserum failed, however, to immunoprecipitate radiolabeled proteins from medium collected from Day 4 blastocysts cultured in the presence of L‐[35S]‐methionine. S1 nuclease analysis of placental RNA and screening of ectoplacental cone and extraembryonic ectoderm cDNA libraries with MuIFN‐α and ‐β probes failed to detect IFN related mRNAs, even under relatively non‐stringent conditions of hybridization. Thus, while antiviral activity is produced by peri‐implantation conceptuses in several diverse mammalian species, it does not appear to be due to a conserved type of IFN in all these species.
Antispectrin antibodies stain the oocyte nucleus and the site of fertilization channels in the egg of Discoglossus pictus (Anura)
Authors: Campanella Chiara , Carotenuto Rosa , Gabbiani G. 
Published: February 2005

AbstractIn Discoglossus pictus eggs, only the dimple contains ionic channels active at fertilization; in particular, chloride channels are found in the central protion of the dimple, which is also the site of sperm penetration. Moreover the dimple hosts an imposing cytoskeleton, consisting of a cortical network and bundles of microfilaments extending from the microvilli. Since spectrin cross links actin and is connected through ankyrin to anion transporters in the plasma membrane of erythrocytes as well as to anion channels in other cells, we studied, in D. pictus egg, the relationship between the localization of spectrin and the high polarization of ionic channels and cytoskeletal organization. By means of immunocytochemistry, we localized spectrin exclusively in the egg dimple. In an attempt to trace back the source of spectrin localization, we immunostained sections of D. pictus ovary and localized spectrin in the nuclei of previtellogenic oocytes, where actin is also present. Antispectrin staining remained until germinal vesicle breakdown. By contrast, a cortical localization was found only when the oocytes divided into two hemispheres and into the germinative area (GA), which, after germinal vesicle breakdown, gives rise to the dimple. At this stage the antispectrin signal was particularly strong in the GA. Using Rho‐pialloidin, we also established that spectrin is generally present where F‐actin is found. However, spectrin and F‐actin do not have the same pattern of fluorescence. In conclusion, our data suggest that spectrin may play a role in oocyte and egg polarity. In eggs, it could be instrumental in anchoring to the cytoskeleton membrane proteins such as receptors and ionic channels, including chloride‐permeable channels.
Effects of cold shock and phospholipase A2 on intact boar spermatozoa and sperm head plasma membranes
Authors: Robertson L. , Bailey J. L. , Buhr M. M. 
Published: February 2005

AbstractHead plasma membranes (HPM) isolated from cryopreserved boar spermatozoa show an excessive fluidization (Buhr et al., Gamete Res 23:441–449, 1989), which might be involved in the loss of fertility. The current study assessed the ability of cold shock (5°C) and phospholipase A2 (PA2) to duplicate these effects on membrane structure and to affect 45Ca2+ uptake and gross morphological characteristics of whole, fresh boar sperm. The HPM from cold‐shocked sperm showed a significantly greater rate of fluidization over time than did HPM from control sperm. Addition of PA2 (bee or snake venom, 0.1 or 10.0 ng/ml) to HPM from control sperm caused fluidization similar to cold shocking, but to a lesser degree (P < 0.05). Cold‐shocked intact sperm exhibited severe acrosomal disruption, loss of motility, and increased 45Ca2+ uptake relative to control sperm. Addition of PA2 (bee or snake venom, 0.1, 1.0., 10.0, and 1,000 ng/ml) to control sperm had not effect on gross morphology or motility while maintaining or increasing sperm extrusion of 45Ca2+. Therefore, although PA2 can, to some extent, duplicate the effects of cold shock on HPM molecular organization, its lipid hydrolytic action is insufficient to cause all the gross disruptions of severe thermal shock. Both PA2 and cold shock disrupted HPM structure, but only cold shock increased 45Ca2+ uptake, suggesting that cold shock may be increasing 45Ca2+ uptake in areas other than the head. Cold shock disrupts sperm on three levels; membrane molecular organization, intracellular Ca2+ regulation, and gross morphology/motility.
Antibodies to sperm surface fertilization antigen (FA‐1): Their specificities and site of interaction with sperm in male genital tract
Authors: Naz R. K. , Bhargava K. K. 
Published: February 2005

AbstractThe fertiization antigen (FA‐1) isolated from murine testes demonstrated its dimeric form of 49,000 ± 2,000 molecular weight (M.W.) or a monomer of 23,000 M.W. on sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE). The FA‐1 was immunogenic in all three female rabbits tested and raised a high‐titer antisera [enzyme‐linked immunosorbent assay (ELISA) titers; 1:1,024 to 1:4,096]. The rabbit anti‐FA‐1 antisera predominantly recognized the dimeric form of 49,000 ± 2,000 M.W. on the Western blot of lithium diiodosalicylate (LIS)‐solubilized murine testes. None of the antisera reacted with any somatic tissue, indicating germ‐cell specificity of FA‐1. To determine the cellular localization of the immunoreactive FA‐1, a novel ultrasensitive immunogold‐silver staining (IGSS) procedure was developed. The anti‐FA‐1‐lgG showed intense staining in the luminal region of the seminiferous tubules containing spermatids and spermatozoa. No reaction was observed in the peripheral area of the tubules containing Sertoli cells, spermatogonia, leptotene, and zygotene spermatocytes. The biodistribution studies of 125I‐labeled anti‐FA‐1 lgG in mice revealed that the antibodies do not bind to somatic tissues such as blood cell, liver, heart, kidney, muscle, and gastrointestinal tissue and do not transudate into testes nad seminal vesicle. However, the antibodies preferentially transudate into epididymis (especially corpus or cauda regions) and vas deferens to bind to sperm cells. In conclusion, our data indicate that FA‐1 can induce an immune response that is germ cell‐specific, directed against later stages of spermatogenesis. The antibodies to FA‐1 interact with sperm after penetration through epididymis (especially corpus and cauda regions) and vas deferens rather than through testes and seminal vesicles.
Changes in protein phosphorylation associated with compaction of the mouse preimplantation embryo
Authors: Bloom Theodora , McConnell Josie 
Published: February 2005

AbstractIn order to investigate the role of protein phosphorylation in the early differentiative events of mouse preimplantation development, timed groups of embryos of various stages were incubated in medium containing [32P]orthophosphate and harvested immediately after labelling or following a chase period. The phosphoproteins obtained were separated by electrophoresis in one and two dimensions. While some of the phosphoproteins found were common to all the stages examined, the detection of many depended on both the combination of pulse‐labelling and chase periods used and on the developmental stage examined. Some phosphoproteins were only found in compacted 8‐cell embryos, a correlation which suggests a possible link with the post‐translational mechanisms which underlie compaction.
Analysis of protein synthesis in morphologically classified bovine follicular oocytes before and after maturation in vitro
Authors: Kastrop P. M. M. , Bevers M. M. , Destrée O. H. J. , Kruip Th. A. M. 
Published: February 2005

AbstractBovine cumulus oocyte complexes (COCs) were isolated from antral ovarian follicles (4–8 mm). Immature COCs were classified into four categories, based on the homogeneity and clearness of the ooplasm and the transparency and compactness of the cumulus investment. In this study, the incorporation of TCA‐precipitable 35S‐methionine and the protein synthesis patterns of oocytes of these four categories were examined. Before maturation in vitro, similar incorporation rates and identical protein synthesis patterns were observed between oocytes of categories 1–3. Immature oocytes of category 4 showed reduced incorporation rates and exhibited aberrant protein synthesis patterns. After maturation in vitro, the patterns of category 4 oocytes were identical with the patterns of those in categories 1–3. The incorporation of 35S‐methionine into in vitro matured oocytes was lower (P < .001) in all categories.Based on these results, it is concluded that the initial classification of oocytes into four categories can be reduced to two categories.
Hyaluronic acid synthesis and gap junction endocytosis are necessary for normal expansion of the cumulus mass
Authors: Chen Lin , Wert Susan E. , Hendrix E. Michael , Russell Paul T. , Cannon Michelle , Larsen William J. 
Published: February 2005

AbstractThe application of a quantitative videographic technique has provided an opportunity to compare the quantitative volumetric expansion of cultured oocyte complexes (COCs) to quantitative changes in gap junction down‐regulation and hyaluronic acid synthesis and to investigate the effects of physiological agents that influence these processes. Results of these experiments support the idea that the down‐regulation of cumulus gap junctions is required for the initial phase of cumulus cell disaggregation and confirm earlier reports that hyaluronic acid synthesis plays a major role in additional expansion of the cumulus. These studies also provide evidence that the degree of expansion observed in culture lacking substrates of hyaluronic synthesis is significantly attentuated when compared with expansion occurring in vivo and that the failure of cultured complexes to expand maximally can be overcome by the addition of substrates of hyaluronic acid synthesis to the culture medium.
Subzonal transfer of multiple sperm (MIST) into early human embryos
Authors: Ng S. C. , Sathananthan A. H. , Bongso T. A. , Ratnam S. S. , Tok V. C. N. , Ho J. K. C. 
Published: February 2005

AbstractMicronsemination sperm transfer (MIST) is a technique whereby sperm are transferred into the perivitelline space (PVS) with the aid of a micromanipulator. MIST is now used to investigate whether blastomere membranes of early human embryos are capable of fusing with the sperm as in the metaphase II oocyte. Between 10 and 30 sperm were transferred into 11 donated human embryos between pronuclear and 16 cell stage. After culture for 6–24 hr in vitro, the embryos were fixed for transmission electron microscopy (TEM). Both acrosome‐intact and acrosome‐reacted sperm were located in the PVS and between blastomeres. Sperm in the PVS were sometimes penetrating the inner regions of the zona. Sperm‐blastomere membrane fusion was not observed, but sperm tail incorporation by phagocytosis was occasionally evident. Sperm heads incorporated into blastomeres were often located in membrane‐bound vesicles. Both acrosome‐intact and acrosome‐reacted sperm heads were found in vacuoles. Acrosome‐reacted sperm heads were lying passively in vacuoles or were undergoing degenerative changes at their surfaces. Sperm chromatin decondensation was not observed in any of the sperm heads that were detected in the blastomeres. The evidence presented clearly shows that sperm heads are incapable of expanding their chromatin to form typical male pronuclei following MIST into early human embryos.
Functions of maternal mRNA in early development
Authors: Dworkin Mark B. , Dworkin‐Rastl Eva 
Published: February 2005

AbstractIn this review, the types of mRNAs found in oocytes and eggs of several animal species, particularly Drosophila, marine invertebrates, frogs, and mice, are described. The roles that proteins derived from these mRNAs play in early development are discussed, and connections between maternally inherited information and embryonic pattern are sought. Comparisons between genetically identified maternally expressed genes in Drosophila and maternal mRNAs biochemically characterized in other species are made when possible. Regulation of the meiotic and early embryonic cell cycles is reviewed, and translational control of maternal mRNA following maturation and/or fertilization is discussed with regard to specific mRNAs.
Maturation‐associated changes in the rat zona pellucida
Authors: Rufas Onit , Shalgi Ruth 
Published: February 2005

AbstractRat follicular oocytes, arrested at prophase I, cannot be fertilized in vitro. This capacity is acquired following resumption of meiosis and a series of changes involving both the oocyte and the cumulus cells surrounding it. Oocytes exposed to sperm at different hours before ovulation show a gradual increase in the permeability of their zona pellucida (ZP). Our study examined whether the ZP, in response to the physiological stimulus for maturation and concomitant with the other oocyte—cumulus components, undergoes maturational changes. Two ZP characteristics were assessed, sensitivity to proteolysis and sperm binding. ZP surrounding oocytes and eggs were collected from five sources: 1) germinal vesicle (GV)‐intact oocytes, 2) preovulatory eggs, 3) ovulated eggs isolated from oviducts of immature females, 4) fertilized eggs, 5) ovulated eggs isolated from oviducts of mature females. All ZP surrounding oocytes/eggs from groups 1–5 were dissolved by trypsin. When solubility by pronase and α‐chymotrypsin was examined, a large variation between groups was found. All ZP from group 2 were dissolved by 0.001% pronase, compared to 0% solubility in group 4. Only 10% of the ZP surrounding GV‐intact oocytes (group 1) were dissolved by this enzyme, compared to 82% in group 3. Solubility in 0.01% α‐chymotrypsin showed a similar pattern. Capacitated sperm were incubated with eggs from groups 1 and 3. The number of sperm binding to ZP in group 3 was repeatedly higher than that in group 1. In both tests it was found that the ZP surrounding the mature eggs differ in their characteristics from ZP of GV‐intact oocytes. These variations may reflect a series of events in the ZP initiated by the GV breakdown and completed with the production of a mature fertilizable egg.
Spermiogenesis in Xenopus laevis: From late spermatids to spermatozoa
Authors: Bernardini Giovanni , Podini Paola , Maci Rosalba , Camatini Marina 
Published: February 2005

AbstractSpermatogenesis is a complex morphogenetic process in which microfilaments and microtubules have been shown to play an important role. The last steps of Xenopus spermatogenesis, i.e., the corkscrew shaping of the sperm head, have been followed to study actin and microtubule distribution by conventional and immunoelectron microscopy. During sperm head morphogenesis, actin is absent in the elongating spermatids, but it is present in the Sertoli cells where results localized at the periphery of their cytoplasm that surrounds the developing germ cells. Sertoli cell actin and microtubules may assist the elongation and the shaping of the spermatids and function in maintaining the Sertoli‐spermatid association.
EGF receptor activities in mammalian development
Authors: Adamson Eileen D. 
Published: February 2005

AbstractThe receptor for epidermal growth factor (EGF) and its analog transforming growth factor alpha (TGFα) is ubiquitous, implying quite general roles for EGF/TGFα in cell viability and tissue maintenance in adult tissues. There is also evidence that the EGF receptor is active in promoting wound healing and tissue regeneration in adult organs, such as skin, liver, and intestinal epithelium. It is likely that EGF receptors have more specific roles during the gestation period. For example, we have detected EGF receptors on the 3.5‐day blastocyst (trophectoderm) surface and since TGFα‐like mRNA sequences and peptides have been detected at this time (Rappolee et al., Science 241:1823, 1988), there is a strong implication for autocrine stimulation in pre‐ and peri‐implantation stage embryos. Paracrine stimulation between the embryo and maternal tissues is also likely since both receptors and TGFα are present in decidual cells. Therefore EGF receptors may take part in growth regulation of the early embryo and in the process of implantation. Other examples where EGF receptors may play specific roles during embryonic development are discussed.
Potential of hypertonic medium treatment for embryo micromanipulation: II. Assessment of nuclear transplantation methodology, isolation, subzona insertion, and electrofusion of blastomeres to intact or functionally enucleated oocytes in rabbits
Authors: Yang X. , Zhang L. , Kovács A. , Tobback C. , Foote R. H. 
Published: February 2005

AbstractThe objective of this research was to study efficiency of embryo development following transfer of blastomeres into the perivitelline space of oocytes. Single blastomeres from 8‐, 16‐, and 32‐cell embryos were obtained following mucin coat and zona pellucida removal by combined treatments with pronase and acidic phosphate‐buffered saline (PBS, pH = 2.5). Blastomeres were separated by pipetting with a fire‐polished micropipette following incubation in Ca++‐free PBS for 15 min at 39°C. This procedure resulted in over 97% blastomere separation. For ease of blastomere insertion, oocytes were placed in droplets of 0.5 M sucrose in PBS (SPBS) during micromanipulation. To functionally enucleate oocytes some were stained with Hoechst 33342 DNA stain and irradiated. A single 8‐ or 16‐cell blastomere was aspirated into an injection pipette (35 μm or 25 μm at the tip, respectively) and inserted into the perivitelline space of an irradiated or non‐irradiated oocyte, but not fused with the oocyte. This micromanipulation procedure did not affect development of individual blastomeres into blastocysts or trophectoderm vesicles when compared with cultured control single blastomeres (P > .05). When the inserted blastomere was induced to fuse with an intact non‐irradiated oocyte under an electric field, 56–57% were fused and 39–45% of the fused and activated oocytes developed to morulae or blastocysts. When an inserted blastomere (from 8–32‐cell embryos) was induced to fuse with a functionally enucleated oocyte treated by Hoechst 33342 staining, followed by washing and UV‐light irradiation, 63–66% of them were fused, but only 15–22% developed to the morula or blastocyst stage. This research demonstrated that the use of hypertonic medium treated oocytes greatly improved the ease and success rate of blastomere subzona insertion, but the value of functionally enucleated oocytes as recipient cells for nuclear transfer requires further investigation.
Effect of microinjection and two types of electrical stimuli on bovine sperm‐hamster egg penetration
Authors: Rickords L. F. , White K. L. , Wiltbank J. N. 
Published: February 2005

AbstractThese experiments were designed to test the effects of an electrofusion and an electroporation pulse on bovine sperm‐hamster egg development. In experiment 1, single motile sperm were injected into the perivitelline space of each egg. A 4,500 V/cm, 30 μsec fusion pulse (FP) was applied while sperm‐egg membrane contact was maintained. It was observed that single motile sperm were rendered immotile immediately after FP application whereas nonpulsed single motile sperm remained motile for up to 36 h postinjection. In addition, both motile and sanicated spermatozoa were injected directly into the ooplasm prior to receiving an FP to determine whether the FP was detrimental to sperm viability. In experiment 2, to induce the acrosome reaction, an 1,150 V/cm electroporation pulse was applied to washed bovine sperm suspended in TALP medium containing 5 mM Ca2+. Treated and nontreated sperm were coincubated with zona‐free hamster ova, and sperm‐penetrating ability was measured. Results from experiment 1 indicate that FP failed to induce sperm‐egg fusion (0/69). FP did not, however, inhibit decondensation or pronuclear formation of sperm injected into hamster egg ooplasm. Single motile sperm injected into the ooplasm resulted in development of both pulsed (19/28) and nonpulsed (21/28) groups. Sonicated tail‐free sperm heads injected into the ooplasm resulted in no detectable difference between treated (18/30) and nontreated (19/30) groups. In experiment 2, treatment of sperm with electroporation pulse +5 mM Ca2+ increased zona‐free hamster ova penetration scores over nontreated sperm within bulls (P < .05). These results indicate that an electroporation pulse in conjunction with high Ca2+ rather than an electrofusion pulse facilitates sperm penetration and early development.
Localization and characterization of the acrosomal antigen recognized by GB24 on human spermatozoa
Authors: Fénichel Patrick , Dohr Gottfried , Grivaux Catherine , Cervoni Florence , Donzeau Michèle , Hsi Bae‐Li 
Published: February 2005

AbstractGB24, a mouse monoclonal antibody, recognizes a trophoblast–leukocyte cross‐reactive antigen (TLX), which is likely identical to the membrane cofactor protein (MCP), a complement regulatory protein. GB24 reacts also with a human acrosomal sperm antigen (Fénichel et al.: J Reprod Fertil 87:699–706, 1989). By immunofluorescence or immunoperoxidase, testicular, epididymal, and ejaculated spermatozoa were found to be positive after fixation by acetone. Motile, suspended spermatozoa became positive only through conditions known to induce acrosome reaction (A23187, follicular fluid, contact with oocytes). Ultrastructural studies with immunogold staining localized this protein on the inner acrosome membrane and in the acrosomal content. By SDS‐polyacrylamide gel electrophoresis, GB24 immunoprecipitated a unique protein of 48 kDa from capacitated and A23187–induced spermatozoa under reducing conditions. No cross‐reactivity was found with mouse, boar, or ram spermatozoa. Localization of this human sperm antigen recognized by GB24 and its similarity with the TLX‐MCP family antigens would suggest a possible role of this molecule during fertilization in sperm–egg binding or immune protection.