pinecone/core-2020-05-10-deduplication huggingface dataset:
Progesterone Triggers a Wave of Increased Free Calcium during the Human Sperm Acrosome Reaction

AbstractThe mammalian sperm acrosome reaction (AR), an essential fertilization event, requires an influx of Ca2+. The Ca2+increase occurring in the human sperm head during the AR initiated by progesterone, a putativein vivoAR initiator, was investigated using video-image analysis with fura-2, a fluorescent Ca2+probe. Progesterone treatment of capacitated human sperm resulted in a wave-like increase in sperm head cytosolic [Ca2+]ithat appears to increase fastest in a region near the equatorial segment and then spreads throughout the rest of the head. The progesterone-mediated Ca2+increase in the sperm head was strongly inhibited and the wave eliminated by picrotoxin, a blocker of GABAAreceptor/Cl−channels and an inhibitor of the progesterone-mediated Cl−efflux and progesterone-initiated AR of human sperm. These results are the first to detect a ligand-mediated Ca2+wave in sperm and to suggest that Cl−efflux influences Ca2+influx during the AR
Stepwise Requirement ofc-kit Tyrosine Kinase in Mouse Ovarian Follicle Development

AbstractOvarian follicle development is controlled by the cycling variation of gonadotrophins derived from the central nervous system. Intragonadal signals are also required, especially in the autonomous development of small follicles. Receptor tyrosine kinasec-kit and its ligand SLF (Steel factor) are expressed on the surface of specific populations of follicle-forming cells in a contiguous manner and are thought to have important roles in follicular development. We blocked the interaction ofc-kit and its ligand by administering the function-blocking antibody ACK2 to developing mice at various times after birth and monitored ovarian follicle development. A blockade ofc-kit function disturbed the onset of primordial follicle development, primary follicle growth, follicular fluid formation of preantral follicles, and penultimate-stage ovarian follicle maturation before ovulation. Ovarian follicle growth was dependent onc-kit during the first 5 days after birth when the functional FSH receptor is not yet expressed in mouse ovary. In contrast, primordial follicle formation and survival, small preantral or antral follicle development, ovulation, and luteinization of the ovulated follicle were not affected by this antibody. These findings indicate the stepwise requirement ofc-kit and its ligand interaction system in the developing ovarian follicle and thatc-kit with its ligand supports the autonomous development of ovarian follicle independent of gonadotrophins
Reexamination of the Sequence of the Sea Urchin Egg Receptor for Sperm: Implications with Respect to Its Properties

AbstractA reexamination of the cDNA clones of theStrongylocentrotus purpuratussea urchin egg receptor for sperm resulted in several important changes to the sequence. By using both rapid amplification of cDNA ends (RACE) and Northern blot analysis for confirmation, the corrected deduced amino acid sequence was shown to lack a classical signal peptide. In addition, a frame shift resulted in a stop codon terminating the deduced sequence prior to a putative transmembrane domain, thereby truncating the protein to 889 amino acids. The deduced amino acid sequence of the receptor has high sequence similarity to the hsp 110 subfamily of proteins. These findings on the primary structure of the egg receptor for sperm raise important questions concerning the mechanism by which the heavily glycosylated receptor is localized to the extracellular surface of the egg and to the cortical granules
Activation of the Meiotic Divisions inDrosophilaOocytes

AbstractKey meiotic events in many organisms are controlled at the translational level. In this study, we examine the role of translational regulation in the meiotic cell cycle ofDrosophila.In order to address this question, we developed a system for activatingDrosophilaoocytesin vitro.With this method, hundreds of mature oocytes can be activated to resume and complete meiosis. The stages of meiosis are normal by cytological criteria, and the timing of the meiotic divisions is similar to that of eggs activatedin vivo.We use this system to examine the role of protein synthesis in regulating the progression of meiosis and the maintenance of the metaphase I arrest. We find that synthesis of new proteins after metaphase I is not required for anaphase I, meiosis II, or the decondensation of the meiotic products. Also, continued protein synthesis is not required to maintain the metaphase I arrest. New protein synthesis is required, however, for proper chromatin recondensation after meiosis
Surface Localization of the Sea Urchin Egg Receptor for Sperm

AbstractThe sea urchin egg receptor for sperm is thought to be involved in species-specific sperm–egg interactions at the egg surface. Recent revisions in the deduced amino acid sequence of the cloned cDNAs indicate that the protein encoded does not possess the common structural hallmarks of a membrane protein. Thus, investigation of the localization and association of the protein with the egg surface is crucial. We describe and characterize a new monoclonal antibody raised against recombinant sperm receptor protein. This antibody, in conjunction with several polyclonal antibodies, was used to study the receptor protein in eggs. Immunoprecipitation studies indicated that the antibodies recognize the highMr(ca. 350 K) sperm receptor protein which copurified with egg plasma membrane–vitelline layer complexes. The sperm receptor protein was solubilized only by detergents and not by treatments designed to solubilize peripherally associated or lipid-anchored membrane proteins, suggesting a tight association with the membrane fraction. Confocal immunofluorescence microscopy of live eggs indicated surface staining. Finally, lysylendoproteinase C treatment of live eggs resulted in a loss of the highMrreceptor protein epitopes, and the concomitant release of a 70-kDa proteolytic fragment, which correlated with a reduced ability of the eggs to be fertilized. Taken together, these data indicate that at least some fraction of the sperm receptor protein is present on the egg surface, a requisite locale for a sperm binding protein
The Organization and Animal–Vegetal Asymmetry of Cytokeratin Filaments in Stage VIXenopusOocytes Is Dependent upon F-Actin and Microtubules

AbstractConfocal immunofluorescence microscopy with anti-cytokeratin antibodies revealed a continuous and polarized network of cytokeratin (CK) filaments in the cortex of stage VIXenopusoocytes. In the animal cortex, CK filaments formed a dense meshwork that both was thicker and exhibited a finer mesh than the network of CK filaments previously observed in the vegetal cortex (Klymkowskyet al.,1987). CK filaments first appeared in association with germinal vesicle (GV) and mitochondrial mass (MM) of oocytes in early mid stage I, indicating that CK filaments are the last of the three cytoskeletal networks to be assembled. By late stage I, CK filaments formed complex networks surrounding the GV, surrounding and penetrating the MM, and linking these networks to a meshwork of CK filaments in the oocyte cortex. During stage III–early IV, CK filaments formed a highly interconnected, apparently unpolarized, radial array linking the perinuclear and cortical CK filament networks. Polarization of the CK filament network was observed during mid stage IV–stage V, as first the animal, then the vegetal CK filament networks adopted the organization characteristic of stage VI oocytes. Treatment of stage VI oocytes with cytochalasin B disrupted the organization of both cortical and cytoplasmic CK filaments, releasing CK filaments from the oocyte cortex and inducing formation of numerous cytoplasmic CK filament aggregates. CB also disrupted the organization of cytoplasmic microtubules (MTs) in stage VI oocytes. Disassembly of oocyte MTs with nocodazole resulted in loss of the characteristic A–V polarity of the cortical CK filament network. In contrast, disruption of cytoplasmic CK filaments by microinjection of anti-CK antibodies had no apparent effect on cytoplasmic or MT organization. We propose a model in which the organization and polarization of the cortical network of CK filaments in stage VIXenopusoocytes are dependent upon a hierarchy of interactions with actin filaments and microtubules
Assessing Normal Embryogenesis inCaenorhabditis elegansUsing a 4D Microscope: Variability of Development and Regional Specification

AbstractCaenorhabditis elegansis renowned for its invariant embryogenesis. This pattern of development is in apparent contrast to other organisms from Drosophila to higher vertebrates. With the aid of a 4D microscope system (multifocal, time-lapse video recording system) which permits the extensive documentation and analysis of cell divisions, cell positions, and migrations in single embryos we have analyzed normal embryogenesis ofC. elegans.The instrumentation reveals a naturally occurring variability in cell division timing, cell positioning, and cell–cell contacts which could not have been detected by the direct observation used earlier (Sulstonet al.,1983,Dev. Biol.100, 64–119). Embryos are very flexible and produce an essentially invariant premorphogenetic stage from variable earlier stages. An analysis of the distribution of the descendants of the early founder blastomeres at the premorphogenetic stage shows that these establish discrete regions in the embryo, a process involving a considerable amount of cell movement, which again varies in different embryos. Only cell fate assignment remains invariant. However, as shown earlier, this is not due to an autonomous invariant specification of cell fates but due to the fact that cell–cell interactions occur very early when the topology of blastomeres in the embryo is still sufficiently precise to ensure reproducible patterns of inductions. A new concept that founder blastomeres produce embryonic regions in the embryo can explain the striking complexity of the lineage per se and also the complicated asymmetric lineage patterns by which the bilateral symmetry of the embryo is established. Many cells, including bilateral homologs, were apparently chosen for a specific fate solely by their position in the embryo, irrespectively of the lineage descent by which the cells are created. We postulate that the production of regions by cell–cell interactions is the pivotal principle guiding the embryogenesis ofC. elegansand that the embryogenesis of the worm follows the same basic principles as embryogenesis in other organisms
The Function ofsilberblickin the Positioning of the Eye Anlage in the Zebrafish Embryo

AbstractIn zebrafish, as in other vertebrates, an initially singular eye field within the neural plate has to split during morphogenesis to allow the development of two separated eyes. It has been suggested that anterior progression of midline tissue within the neural plate is involved in the bilateralization of the eye field. Mutations in the recently identifiedsilberblick(slb) gene cause an incomplete separation of the eyes. During gastrulation and early somitogenesis, the ventral midline of the central nervous system (CNS) together with the underlying axial mesendoderm is shortened and broadened inslbembryos. While in wild-type embryos the ventral CNS midline extends to the anterior limit of the neural plate at the end of gastrulation, there is a gap between the anterior tip of the ventral CNS midline and the anterior edge of the neural plate inslb.To investigate the cause for the shortening of the ventral CNS midline inslbwe determined the fate of labeled ventral CNS midline cells in wild-type andslbembryos at different stages of development. Inslb,anterior migration of ventral CNS midline cells is impaired, which indicates that migration of these cells is needed for elongation of the ventral CNS midline. The anterior shortening of the ventral CNS midline inslbleads to medial instead of bilateral induction of optic stalks followed by a partial fusion of the eyes at later developmental stages. The analysis of theslbphenotype indicates that anterior migration of midline cells within the neural plate is required for proper induction and subsequent bilateralization of an initially singular eye field. These findings may therefore provide a starting point in elucidating the role of neural plate morphogenesis in positioning of the eyes
Oxygen Regulates Vascular Endothelial Growth Factor-Mediated Vasculogenesis and Tubulogenesis

AbstractTo determine whether low oxygen is a stimulus for endothelial cell differentiation and vascular development in the kidney, we examined the effect of low oxygen on rat metanephric organ culture, a model known to recapitulate nephrogenesis in the absence of vessels. After 6 days in culture in standard (20% O2) or low oxygen (1–3% O2) conditions, metanephric kidney growth and morphology were assessed by DNA measurement, and light and electron microscopy. DNA content was higher in 3% O2-treated explants (2.5 ± 0.17 μg/kidney,n= 9) than in 20% O2explants (1.5 ± 0.09 μg/kidney,n= 9),P< 0.05. Low oxygen induced proliferation of tubular epithelial cells, resulting in enhanced number of tubules of similar size. Endothelial cells forming capillaries were localized in 3% O2explants by light and electron microscopy and by immunocytochemistry using endothelial cell markers. Flt-1, Flk-1, and ACE-containing cells were detected in 3% O2-treated explants, whereas 20% O2explants were virtually negative. VEGF mRNA levels were 10-fold higher in 3% O2-treated explants than in 20% O2-treated explants. Addition of anti-VEGF antibodies to 3% O2-treated explants prevented low oxygen-induced growth and endothelial cell differentiation and proliferation. Our data indicate that low oxygen stimulates growth by cell proliferation and induces tubulogenesis, endothelial cell differentiation, and vasculogenesis in metanephric kidneys in culture. Upregulation of VEGF expression by low oxygen and prevention of low oxygen-induced tubulogenesis and vasculogenesis by anti-VEGF antibodies indicate that these changes were mediated by VEGF. These data suggest that low oxygen is the stimulus to initiate renal vascularization
Developmental Testis-Specific Regulation of mRNA Levels and mRNA Translational Efficiencies for TATA-Binding Protein mRNA Isoforms

AbstractEarly spermatids contain roughly 1000-fold more TATA-binding protein (TBP) mRNA than do somatic cells. The appearance of TBP-overexpressing spermatids in the developing testis is accompanied by a large increase in whole-organ levels of total RNA and of poly(A)+RNA per cell. Whereas somatic cells initiate transcription of TBP mRNA at a single promoter/first exon (exon 1C), in adult testis, two additional major promoter/first exons (1D and 1E) are used. We have examined the expression of the somatic and testis-specific TBP mRNA isoforms during rodent testis development. In juvenile testes TBP mRNAs containing either exon 1C or exon 1D, but none containing exon 1E, are detected. At 21 days of age, all TBP mRNA isoforms begin to overaccumulate. The onset of TBP mRNA overaccumulation is marked first by an increase in levels of polysomal TBP mRNA, and later by accumulation of mRNP-associated TBP mRNA. In adult testes, only 30% of the total TBP mRNA is engaged by polysomes; the remainder is sequestered as mRNP particles. All of the TBP mRNA isoforms in adults exist both as free mRNP particles and as polysomes; however, the fraction in polysomes varies from 60% (exon 1C) to 10% (exon 1E). This suggests that sequences within the first exons alter the probability that the mRNA will either assemble into polysomes or into translationally inactive mRNP particles
Spatial Regulation of Neuronal Gene Expression in Response to Nerve Growth Factor

AbstractTo examine the cellular mechanisms whereby distally derived growth factors regulate nuclear responses in neurons, we have utilized compartmented cultures of sympathetic neurons to examine the regulation of two nerve growth factor (NGF)-inducible genes, tyrosine hydroxylase (TH) and p75 neurotrophin receptor (p75NTR). These studies demonstrate that NGF can signal retrogradely to mediate the induction of TH and p75NTR mRNAs. However, quantitative differences occurred as a function of the spatial localization of NGF exposure; application of NGF to cell bodies and proximal axons elicited peak levels of neuronal gene expression that were two- to threefold higher than when NGF was applied to distal axons alone. Furthermore, neurons responding maximally to NGF on distal axons were still able to respond to NGF administered to cell bodies and proximal axons. Biochemical analysis indicated that this difference in responsiveness was not due to differences in the number of TrkA/NGF receptors in the two compartments. Thus, although NGF signals retrogradely to mediate nuclear responses, the magnitude of these responses differs as a function of the spatial location of the activated NGF receptor:ligand complex. Moreover, these data suggest that neurons may be able to respond to a second cellular source of neurotrophins, even when target-derived neurotrophins are not limiting
C. elegansCell Migration Genemig-10Shares Similarities with a Family of SH2 Domain Proteins and Acts Cell Nonautonomously in Excretory Canal Development

AbstractThemig-10gene ofCaenorhabditis elegansis required for the long-range anteroposterior migration of embryonic neurons CAN, ALM, and HSN and proper development of the excretory canals. Here, we report the cloning and initial molecular characterization ofmig-10.The predicted MIG-10 proteins share a large region of similarity with a recently identified family of mammalian SH2 domain proteins, Grb7 and Grb10. We call this region of similarity the GM region (for Grb and Mig). MIG-10 proteins do not contain an SH2 domain, but share with the Grbs a pleckstrin homology (PH) domain and proline-rich regions, features commonly found in signal transduction proteins. The functions of Grb7 and Grb10 are unknown, but Grb7 is overexpressed in certain breast cancers, where it is bound to the growth factor receptor HER2, while Grb10 has been implicated in insulin signaling. We also report the isolation of a newmig-10(e2527)allele, as well as the molecular characterization ofe2527(splice acceptor mutation) and the canonicalct41(amber) allele. Finally, we report the results of a genetic mosaic analysis which reveal thatmig-10acts cell nonautonomously in the development of the excretory canals and suggest a possible focus formig-10activity within descendants of the AB cell lineage. Elucidation of the role ofmig-10inC. elegansdevelopment should lead to a better understanding of cell migration and may shed light on the function of a family of SH2 domain proteins apparently involved in signal transduction and cancer
Stage-Specific Signals in Germ Line Differentiation: Control of Sertoli Cell Phagocytic Activity by Spermatogenic Cells

AbstractDifferentiation of male germ cells requires a continuous cross-talk with their somatic support, the Sertoli cell. Anin vitromodel of Sertoli cells was recently provided by established cell lines which maintain Sertoli-specific characteristics, among which is a regulated phagocytic capacity.In vivo,Sertoli cells take up the residual cytoplasm expelled from the maturing sperm, a process restricted to a limited period of germinal maturation, and they also eliminate abnormally differentiated germ cells in case of hormonal deficiency. Cells of the Sertoli line efficiently take up latex beads, as well as dead cells in the cultures. A semiquantitative assay of phagocytosis was developed, based on the uptake of fluorescent latex beads. 15P-1 cultures were found to contain a minor fraction of active phagocytes. After addition of a defined fraction of germ cells, however, all the cells internalized beads as efficiently as macrophages. The inducing cell was identified as the pachytene spermatocyte, a cell type which,in vivo,is associated with Sertoli cells when they express their phagocytic potential. These inducing meiotic cells were not internalized themselves. Rather, they interacted with Sertoli cells via a surface signal that was resistant to formaldehyde fixation. The whole induction process does not involve changes in Sertoli gene expression, since it occurs even in the presence of high doses of cycloheximide. After the required initial contact, further maintenance of the activity was dependent on factor(s) secreted in the medium of the activated culture. Phagocytosis was, on the other hand, abrogated in the presence of factor(s) secreted by a distinct fraction of germ cells, enriched in the late stages (second division) of meiosis
Retinoic Acid Disturbs Mouse Middle Ear Development in a Stage-Dependent Fashion

AbstractThe mammalian middle ear contains a chain of three ossicles, the malleus, incus, and stapes, that transmit into the inner ear the vibrations produced in the tympanic membrane by aerial sound. I show here that retinoic acid interferes with the formation of the middle ear in a stage-specific fashion. The malformations produced are derived from a retinoic acid-induced inhibition of the formation and/or migration of the cranial neural crest that generates the middle ear skeletal elements and not from a respecification of neural crest identity to more posterior fates. I have used these effects of retinoic acid to analyze the temporal sequence of events involved in the morphogenesis of the middle ear. I show that the middle ear bones develop from several primordia that originate in a typical temporal sequence from Day 8 plus 4.5 hr to Day 8 plus 7.5 hr of pregnancy. Moreover, interactions between adjacent bones are not required for their proper formation. My results also suggest aHoxa-2-mediated patterning of the otic capsule in the region where the oval window is located
Autocrine Regulation of Neural Crest Cell Development by Steel Factor

AbstractSteel factor (SLF) and its cognate receptor, c-kit, have been implicated in the generation of melanocytes from migrating neural crest (NC) cells during early vertebrate embryogenesis. However, the source of SLF in the early avian embryo and its precise role in melanogenesis are unclear. We report here that NC cells themselves express and release SLF protein, which in turn acts as an autocrine factor to induce melanogenesis in nearby NC cells. These results indicate that NC cell subpopulations play an active role in the determination of their cell fate and suggest a different developmental role for the embryonic microenvironment than what has been previously proposed