pinecone/core-2020-05-10-deduplication huggingface dataset:
Localization of a Novel Human A-Kinase-Anchoring Protein, hAKAP220, during Spermatogenesis

AbstractUsing a combination of protein kinase A type II overlay screening, rapid amplification of cDNA ends, and database searches, a contig of 9923 bp was assembled and characterized in which the open reading frame encoded a 1901-amino-acid A-kinase-anchoring protein (AKAP) with an apparent SDS–PAGE mobility of 220 kDa, named human AKAP220 (hAKAP220). The hAKAP220 amino acid sequence revealed high similarity to rat AKAP220 in the 1167 C-terminal residues, but contained 727 residues in the N-terminus not present in the reported rat AKAP220 sequence. The hAKAP220 mRNA was expressed at high levels in human testis and in isolated human pachytene spermatocytes and round spermatids. The hAKAP220 protein was present in human male germ cells and mature sperm. Immunofluorescent labeling with specific antibodies indicated that hAKAP220 was localized in the cytoplasm of premeiotic pachytene spermatocytes and in the centrosome of developing postmeiotic germ cells, while a midpiece/centrosome localization was found in elongating spermatocytes and mature sperm. The hAKAP220 protein together with a fraction of PKA types I and II and protein phosphatase I was resistant to detergent extraction of sperm tails, suggesting an association with cytoskeletal structures. In contrast, S-AKAP84/D-AKAP1, which is also present in the midpiece, was extracted under the same conditions. Anti-hAKAP220 antisera coimmunoprecipitated both type I and type II regulatory subunits of PKA in human testis lysates, indicating that hAKAP220 interacts with both classes of R subunits, either through separate or through a common binding motif(s)
MAP Kinase, a Universal Suppressor of Sperm Centrosomes during Meiosis?

AbstractWe reported previously that inhibition of MAP kinase during meiosis in Urechis caupo eggs caused premature sperm aster formation and we reviewed indirect evidence that the suppression of sperm asters by MAPK during meiosis might be a universal mechanism (M. C. Gould and J. L. Stephano, 1999, Dev. Biol. 216, 348–358). We tested this proposition with oyster (Crassostrea gigas) and starfish (Asterina miniata) eggs, utilizing the MEK inhibitors U0126 and PD98059. Centrosomes, asters, and meiotic spindles were visualized by normal epifluorescence and confocal microscopy following indirect immunocytochemical staining for anti-β-tubulin. When MAPK activation was inhibited, sperm asters in both species developed prematurely and tended to move toward the egg centrosomes, sometimes even fusing with the egg spindle or centrosomes. Meiotic spindles and polar body formation were also abnormal when MAPK was inhibited
Distinct Mechanisms Triggering Glial Differentiation in Drosophila Thoracic and Abdominal Neuroblasts 6-4

AbstractNeurons and glia are produced in stereotyped patterns after neuroblast cell division during development of the Drosophila central nervous system. The first cell division of thoracic neuroblast 6-4 (NB6-4T) is asymmetric, giving rise to a glial precursor cell and a neuronal precursor cell. In contrast, abdominal NB6-4 (NB6-4A) divides symmetrically to produce two glial cells. To understand the relationship between cell division and glia–neuron cell fate determination, we examined and compared the effects of known cell division mutations on the NB6-4T and NB6-4A lineages. Based on observation of expression of glial fate determination and early glial differentiation genes, the onset of glial differentiation occurred in NB6-4A but not in NB6-4T when both cell cycle progression and cytokinesis were genetically arrested. On the other hand, glial differentiation started in both lineages when cytokinesis was blocked with intact cell cycle progression. These results showed that NB6-4T, but not NB6-4A, requires cell cycle progression for acquisition of glial fate, suggesting that distinct mechanisms trigger glial differentiation in the different lineages
Inositol 1,4,5-Trisphosphate Receptors Are Downregulated in Mouse Oocytes in Response to Sperm or Adenophostin A but Not to Increases in Intracellular Ca2+ or Egg Activation

AbstractFertilization in mammals stimulates a series of Ca2+ oscillations that continue for 3–4 h. Cell-cycle-dependent changes in the ability to release Ca2+ are one mechanism that leads to the inhibition of Ca2+ transients after fertilization. The downregulation of InsP3Rs at fertilization may be an additional mechanism for inhibiting Ca2+ transients. In the present study we examine the mechanism of this InsP3R downregulation. We find that neither egg activation nor Ca2+ transients are necessary or sufficient for the stimulation of InsP3R downregulation. First, parthenogenetic activation fails to stimulate downregulation. Second, downregulation persists when fertilization-induced Ca2+ transients and egg activation are inhibited using BAPTA. Third, downregulation can be induced in immature oocytes that do not undergo egg activation. Other than fertilization, the only stimulus that downregulated InsP3Rs was microinjection of the potent InsP3R agonist adenophostin A. InsP3R downregulation was inhibited by the cysteine protease inhibitor ALLN but MG132 and lactacystin were not effective. Finally, we have injected maturing oocytes with adenophostin A and produced MII eggs depleted of InsP3Rs. We show that sperm-induced Ca2+ signaling is inhibited in such InsP3R-depleted eggs. These data show that InsP3R binding is sufficient for downregulation and that Ca2+ signaling at fertilization is mediated via the InsP3R
Biphasic Elevation of [Ca2+]i in Individual Human Spermatozoa Exposed to Progesterone

AbstractFluorimetric studies on progesterone-induced [Ca2+]i signalling in mammalian spermatozoa show both the well-characterised [Ca2+]i transient and a subsequent sustained phase. However, the sustained phase is thought to reflect release of the fluorochrome during the acrosome reaction and has not been subject to critical investigation. We have used single-cell imaging of [Ca2+]i to analyse the progesterone-induced [Ca2+]i response in large numbers (>2000) of capacitated, human spermatozoa. In 70% of cells, treatment with progesterone induced a transient increase, which typically peaked within 1 min and decayed with a similar time course. Upon rapid application of progesterone this response peaked within 5–20 s. In 35% of progesterone-treated spermatozoa a sustained elevation of [Ca2+]i occurred, which became discernible during the falling phase of the transient response and persisted for at least 20 min. Both [Ca2+]i responses were localised to the postacrosomal region. Averaging of large numbers of single cell responses generated traces similar to those seen in fluorimetric studies. Although the sustained response was strongly associated with the initial, transient response, a few spermatozoa generated sustained responses that were not preceded by a significant transient response (5% of cells). It is concluded that a genuine biphasic [Ca2+]i signal is activated by progesterone and that the sustained response is a discrete signalling event with biological significance
Role for cGATA-5 in Transcriptional Regulation of the Embryonic Chicken Pepsinogen Gene by Epithelial–Mesenchymal Interactions in the Developing Chicken Stomach

AbstractA gene encoding embryonic chicken pepsinogen (ECPg), a zymogen of the digestive enzyme pepsin, is expressed specifically in epithelial cells of glands of embryonic stage proventriculus (glandular stomach) under the influence of mesenchyme. We found four GATA and one Sox binding motifs in 1.1 kb of the 5′ flanking region of the ECPg gene which are essential to the organ-specific expression of the gene. The expression of cGATA-5 and cSox2 in the proventriculus from day 6 to day 12 of incubation was therefore analyzed. cGATA-5 was more strongly expressed in glandular epithelial cells than in luminal epithelial cells, while cSox2 gene expression was weaker in glandular epithelial cells. Using heterologous recombination explants we also discovered that the expression of cGATA-5 and cSox2 in epithelial cells was affected by mesenchyme when the latter induced ECPg gene expression in epithelial cells. Introduction of expression constructs into epithelial cells by electroporation demonstrated that cGATA-5 upregulated transcription of a reporter luciferase gene via a cis element in the 5′ flanking region of the ECPg gene. The gel mobility shift assay revealed that the cGATA-5 protein specifically binds to the GATA binding sites. cSox2 downregulated the activity of luciferase but it was not through the Sox binding motif. These results suggest that cGATA-5 positively regulates transcription of the ECPg gene and is involved in spatial regulation of the pepsinogen gene during development
Targeting of cre to the Foxg1 (BF-1) Locus Mediates loxP Recombination in the Telencephalon and Other Developing Head Structures

AbstractThe use of genetics to study the development of the telencephalon and derivatives such as the cerebral cortex has been limited. The telencephalon begins to form midway through gestation, and targeted mutations in genes suspected of playing roles in its development often lead to early phenotypes that preclude analysis of their role at later stages. This problem can be circumvented using a Cre/loxP recombination system. A mouse line was produced in which cre was targeted to the Foxg1 (BF-1) locus, a gene expressed specifically in the telencephalon and discrete head structures. Crosses between Foxg1–Cre mice and three separate loxP reporter mice generated embryos with recombination patterns matching that expected from the normal pattern of Foxg1 expression. Recombination occurs invariably in the telencephalon, anterior optic vesicle, otic vesicle, facial and head ectoderm, olfactory epithelium, mid–hindbrain junction, and pharyngeal pouches. Recombination in some animals also occurs less efficiently in tissues not known to express Foxg1. We show that the genetic background of the parental mice and the loxP target allele can each contribute to differences in the exact pattern of recombination. Collectively, these data show that Foxg1–Cre mice should be useful in the deletion or ectopic expression of any floxed target gene in a Foxg1-like pattern
Lim1 Activity Is Required for Intermediate Mesoderm Differentiation in the Mouse Embryo

AbstractDuring gastrulation and early organogenesis, Lim1 is expressed in the visceral endoderm, the anterior mesendoderm, and the lateral mesoderm that comprises the lateral plate and intermediate mesoderm. A previous study has reported that kidneys and gonads are missing in the Lim1 null mutants (W. Shawlot and R. R. Behringer, 1995, Nature 374, 425–430). Results of the present study show that in the early organogenesis stage mutant embryo, the intermediate mesoderm that contains the urogenital precursor tissues is disorganized and displays diminished expression of PAX2 and the Hoxb6-lacZ transgene. When posterior epiblast cells of the Lim1 null mutant embryo were transplanted to the primitive streak of wild-type host embryos, they were able to colonize the lateral plate and intermediate mesoderm of the host, suggesting that Lim1 activity is not essential for the allocation of epiblast cells to these mesodermal lineages. However, most of the mutant cells that colonized the lateral and intermediate mesoderm of the host embryo did not express the Hoxb6-lacZ transgene, except for some cells that were derived from the distal part of the posterior epiblast. Lim1 activity may therefore be required for the full expression of this transgene that normally marks the differentiation of the lateral plate and intermediate mesoderm
Real-Time Measurements of the Interactions between Fluorescent Speract and Its Sperm Receptor

AbstractLytechinus pictus sea urchin sperm express receptors for speract, a sperm-activating peptide derived from the homologous egg jelly coat. We found that the fluorescence of fluorophore-labeled, active, speract analogs is quenched upon receptor binding. This property allowed us to perform real-time measurements of speract–receptor interactions using intact sperm and to determine, for the first time, their association (kon) and dissociation (koff) rate constants. The high kon (2.4 × 107 M−1 s−1) and low koff (4.4 × 10−6 s−1 (95%) and 3.7 × 10−4 s−1 (5%)) can account for the sperm response to picomolar concentrations of speract. We also examined the influence of extracellular ions on speract–receptor interactions using the fluorescence quenching method described in this study. The association rate of speract to the receptor is dramatically reduced in Na+-free seawater (NaFSW), divalent cation-free seawater (DCFSW), and high-K+ seawater (HKSW). In seawater speract induces an increase in intracellular pH (pHi), while it is unable to do so in either NaFSW or HKSW. To test if the lack of this pHi change causes the reduction in the speract association rate, pHi was increased with NH4Cl (10 mM) at the time labeled speract was added. Interestingly, this procedure completely (in HKSW) or partially (in NaFSW and DCFSW) restored the speract association rate to its receptor. These findings indicate that an increase in sperm pHi positively affects the receptor binding activity for this peptide and may partially explain the positive binding cooperativity displayed by the speract receptor
Distribution of Dorsal-Forming Activity in Precleavage Embryos of the Japanese Newt, Cynops pyrrhogaster: Effects of Deletion of Vegetal Cytoplasm, UV Irradiation, and Lithium Treatment

AbstractTwo types of axis-deficient embryos developed after deletion of the vegetal cytoplasm: wasp-shaped embryos and permanent-blastula-type embryos. In situ hybridization revealed that neither type of axis-deficient embryo expressed goosecoid or pax-6. brachyury was expressed in the constricted waist region of the wasp-shaped embryos but was not expressed in the permanent-blastula-type embryos. Further, we examined the effect of UV irradiation on Japanese newt embryos. Surprisingly, UV-irradiated Japanese newt eggs formed hyperdorsalized embryos. These embryos gastrulated in an irregular circular fashion with goosecoid expression in the circular equatorial region. At tailbud stage, these embryos formed a proboscis which is very reminiscent of that formed in hyperdorsalized Xenopus embryos. Transplantation of the marginal region of the UV-irradiated embryos revealed that the entire marginal zone had organizer activity. Thus we conclude that UV hyperdorsalizes Japanese newt embryos. Finally, lithium treatment of normal embryos at the 32-cell stage also resulted in hyperdorsalization. Lithium treatment of vegetally deleted embryos had two distinct results. Lithium treatment of permanent-blastula-type embryos did not result in the formation of dorsal axial structures, while the same treatment reinduced gastrulation and dorsal axis formation in the wasp-shaped embryos. Based on these results, we propose a model for early axis specification in Japanese newt embryos. The model presented here is fundamentally identical to the Xenopus model, with some important modifications. The vegetally located determinants required for dorsal development (dorsal determinants, DDs) are distributed over a wider region at fertilization in Japanese newt embryos than in Xenopus embryos. The marginal region of the Japanese newt embryo at the beginning of development overlaps with the field of the DDs. Gastrulation is very likely to be a dorsal marginal-specific property, while self-constriction is most probably a ventral marginal-specific property in Japanese newt embryos
Developmental Changes in the Spatial Expression of Genes Involved in Myosin Function in Dictyostelium

AbstractWe analyzed the spatial expression patterns of the genes involved in myosin function by in situ hybridization at the tipped aggregate and early culmination stages of Dictyostelium. Myosin heavy chain II mRNA was enriched in the anterior prestalk region of the tipped aggregates, whereas it disappeared from there and began to appear in both upper and lower cups of the early culminants. Similarly, mRNAs for essential light chain, regulatory light chain, myosin light chain kinase A, and myosin heavy chain kinase C were enriched in the prestalk region of the tipped aggregates. However, expression of these genes was distinctively regulated in the early culminants. These findings suggest the existence of mechanisms responsible for the expression of particular genes
Regulation of Gut and Heart Left–Right Asymmetry by Context-Dependent Interactions between Xenopus Lefty and BMP4 Signaling

AbstractThe Lefty subfamily of TGFβ signaling molecules has been implicated in early development in mouse, zebrafish, and chick. Here, we show that Xenopus lefty (Xlefty) is expressed both bilaterally in symmetric midline domains and unilaterally in left lateral plate mesoderm and anterior dorsal endoderm. To examine the roles of Xlefty in left–right development, we created a system for scoring gut asymmetry and examined the effects of unilateral Xlefty misexpression on gut development, heart development, and Xnr-1 and XPitx2 expression. In contrast to the unilateral effects of Vg1, Activin, Nodal, or BMPs, targeted expression of Xlefty in either the left or the right side of Xenopus embryos randomized the direction of heart looping, gut coiling, and left–right positioning of the gut and downregulated the asymmetric expression of Xnr-1 and XPitx2. It is currently thought that Lefty proteins act as feedback inhibitors of Nodal signaling. However, this would not explain the effects of right-sided Xlefty misexpression. Here, we show that Xlefty interacts with the signaling pathways of other members of the TGFβ family during left–right development. Results from coexpression of Xlefty and Vg1 indicate that Xlefty can nullify the effects of Vg1 ectopic expression and that Xlefty is downstream of left-sided Vg1 signaling. Results from coexpression of Xlefty and XBMP4 indicate that XLefty and XBMP4 interact both synergistically and antagonistically in a context-dependent manner. We propose a model in which interactions of Xlefty with multiple members of the TGFβ family enhance the differences between the right-sided BMP/ALK2/Smad pathway and the left-sided Vg1/anti-BMP/Nodal pathway, leading to left–right morphogenesis of the gut and heart
Is Chordin a Long-Range- or Short-Range-Acting Factor? Roles for BMP1-Related Metalloproteases in Chordin and BMP4 Autofeedback Loop Regulation

AbstractDiffusible morphogen models have been used widely to explain regional specification of tissues and body axes during animal development. The three-signal model for patterning the dorsal–ventral axis of the amphibian embryo proposes, in part, that a factor(s) secreted from Spemann's organizer is responsible for converting lateral marginal zone into more dorsal cell fates. We examine the possibility that chordin, a secreted inhibitor of bone morphogenetic protein (BMP) signaling and candidate “dorsalizing signal,” is a long-range-acting factor. We show that chordin can, when overexpressed, act directly over distances of at least 450 μm in the early Xenopus embryo to create a gradient of BMP signaling. However, since lower levels of chordin can still induce secondary axes and these amounts of chordin act only locally to inhibit a BMP target gene, we suggest that chordin likely acts as a short-range signal in vivo. Furthermore, BMP1, a secreted metalloprotease that cleaves chordin protein in vitro, inhibits chordin's axis-inducing effects, suggesting that BMP1 functions to negatively regulate chordin's action in vivo. A dominant-negative mutant BMP1 blocks the in vitro cleavage of chordin protein by wild-type BMP1 and induces secondary axes when injected ventrally. We argue that BMP1 and Xolloid are probably functionally redundant metalloproteases and may have two roles in the early Xenopus embryo. One role may be to inhibit the action of low-level chordin protein expressed throughout the entire embryo and a possible second role may be to inhibit activation of a juxtacrine cell relay, thereby confining chordin's action to the organizer region preventing chordin from functioning as a long-range-acting factor
Gpbox (Psx2), a Homeobox Gene Preferentially Expressed in Female Germ Cells at the Onset of Sexual Dimorphism in Mice

AbstractXX gonads differentiate into ovaries, a morphologic event evident by embryonic day 13.5 (E13.5) in mice. To identify early markers of oogenesis, sex-specific urogenital ridge cDNA libraries were constructed from E12–13 embryos. After mass excision and isolation of plasmid DNA, approximately 4800 expressed sequence tags were determined and compared to existing databases. Few cDNAs were specifically expressed in the urogenital ridge, but one, designated GPBOX, encodes a 227-amino-acid homeobox protein that is first expressed at E10.5 in the embryo as well as in the extraembryonic tissues. The Gpbox gene is single copy in the mouse genome and is located on the X chromosome in close proximity to two other homeobox genes, Pem and Psx1. Within the embryo, its expression is limited to the gonad, and transcripts are not detected in adult tissues. Although comparable levels are initially present in both sexes, GPBOX transcripts accumulate faster in female germ cells and peak at E12.5 when they are present in fivefold greater abundance than in males. The persistence of GPBOX transcripts in female germ cells until E15.5 and their virtual disappearance in males by E13.5 suggest that Gpbox may play a role in mammalian oogenesis
Calcium Influx Mediates the Voltage-Dependence of Sperm Entry into Sea Urchin Eggs

AbstractSperm entry was monitored in voltage-clamped sea urchin eggs following insemination in a variety of artificial seawaters. In regular seawater, maintaining the membrane potential at increasingly negative values progressively inhibits sperm entry. Reducing [Ca2+]o relieves the inhibition, shifting the sperm entry vs voltage relationship toward more negative potentials. Raising [Ca2+]o shifts the relationship in the other direction. Large changes in [Na+]o or [Mg2+]o do not affect sperm entry although changing [Na+]o dramatically changes the currents following sperm attachment. Applying one of seven different calcium channel blockers or replacing Ca2+ with Ba2+ or Sr2+ or microinjecting calcium chelators into the cytoplasm relieves the block to sperm entry at negative potentials. We conclude that the block to sperm entry at negative potentials is mediated by calcium which crosses the membrane and acts at an intracellular site
Importin-α3 Is Required at Multiple Stages of Drosophila Development and Has a Role in the Completion of Oogenesis

AbstractThe Drosophila importin-α3 gene was isolated through its interaction with the large subunit of the DNA polymerase α in a two-hybrid screen. The predicted protein sequence of Importin-α3 is 65–66% identical to those of the human and mouse importin-α3 and α4 and 42.7% identical to that of Importin-α2 (Oho31/Pendulin), the previously reported Drosophila homologue. Both Importin-α3 and Importin-α2 interact with similar subsets of proteins in vitro, one of which is Ketel, the importin-β homologue of Drosophila. importin-α3 is an essential gene, whose encoded protein is expressed throughout development. During early embryogenesis, Importin-α3 accumulates at the nuclear membrane of cleavage nuclei, whereas after blastoderm formation it is characteristically found within the interphase nuclei. Nuclear localisation is seen in several tissues throughout subsequent development. During oogenesis its concentration within the nurse cell nuclei increases during stages 7–10, concomitant with a decline in levels in the oocyte nucleus. Mutation of importin-α3 results in lethality throughout pupal development. Surviving females are sterile and show arrest of oogenesis at stages 7–10. Thus, Importin-α3-mediated nuclear transport is essential for completion of oogenesis and becomes limiting during pupal development. Since they have different expression patterns and subcellular localisation profiles, we suggest that the two importin-α homologues are not redundant in the context of normal Drosophila development