scholarly journals Fyn kinase is involved in cleavage furrow ingression during meiosis and mitosis

Reproduction ◽  
2010 ◽  
Vol 140 (6) ◽  
pp. 827-834 ◽  
Author(s):  
Mattan Levi ◽  
Bernard Maro ◽  
Ruth Shalgi
Keyword(s):  
Cell Cycle Control ◽  
Polar Body ◽  
Mitotic Division ◽  
Dominant Negative ◽  
Confocal Imaging ◽  
Cleavage Furrow ◽  
Filamentous Actin ◽  
Fyn Kinase ◽  
Second Polar Body ◽  
First Time

Fertilization of mammalian oocytes triggers their exit from the second meiotic division metaphase arrest. The extrusion of the second polar body (PBII) that marks the completion of meiosis is followed by the first mitotic cleavage of the zygote. Several lines of evidence in somatic cells imply the involvement of Fyn, an Src family kinase (SFK), in cell cycle control and actin functions. In this study, we demonstrate, using live cell confocal imaging and microinjection of Fyn cRNAs, the recruitment of Fyn to the oocyte's cortical area overlying the chromosomes and its colocalization with filamentous actin (F-actin) during exit from the meiotic metaphase. Fyn concentrated asymmetrically at the cortical site designated for ingression of the PBII cleavage furrow, where F-actin had already been accumulated, and then redispersed throughout the entire cortex only to be recruited again to the cleavage furrow during the first mitotic division. Although microinjection of dominant negative Fyn did not affect initiation of the cleavage furrow, it prolonged the average duration of ingression, decreased the rates of PB extrusion and of the first cleavage, and led to the formation of bigger PBs and longer spindles. Extrusion of the PBII was blocked in oocytes exposed to SU6656, an SFK inhibitor. Our results demonstrate, for the first time, a continuous colocalization of Fyn and F-actin during meiosis and imply a role for the SFKs, in general, and for Fyn, in particular, in regulating pathways that involve actin cytoskeleton, during ingression of the meiotic and mitotic cleavage furrows.

The conformation and activation of Fyn kinase in the oocyte determine its localisation to the spindle poles and cleavage furrow

2011 ◽  
Vol 23 (7) ◽  
pp. 846 ◽  
Author(s):  
Mattan Levi ◽  
Bernard Maro ◽  
Ruth Shalgi
Keyword(s):  
Cell Cycle Control ◽  
Polar Body ◽  
Active Form ◽  
Spindle Pole ◽  
Confocal Imaging ◽  
Cleavage Furrow ◽  
Membrane Area ◽  
Spindle Poles ◽  
N Terminus ◽  
And Function

Several lines of evidence imply the involvement of Fyn, a Src family kinase, in cell-cycle control and cytoskeleton organisation in somatic cells. By live cell confocal imaging of immunostained or cRNA-microinjected mouse oocytes at metaphase of the second meiotic division, membrane localisation of active and non-active Fyn was demonstrated. However, Fyn with a disrupted membrane-binding domain at its N-terminus was targeted to the cytoplasm and spindle in its non-active form and concentrated at the spindle poles when active. During metaphase exit, the amount of phosphorylated Fyn and of spindle-poles Fyn decreased and it started appearing at the membrane area of the cleavage furrow surrounding the spindle midzone, either asymmetrically during polar body II extrusion or symmetrically during mitosis. These results demonstrate that post-translational modifications of Fyn, probably palmitoylation, determine its localisation and function; localisation of de-palmitoylated active Fyn to the spindle poles is involved in spindle pole integrity during metaphase, whereas the localisation of N-terminus palmitoylated Fyn at the membrane near the cleavage furrow indicates its participation in furrow ingression during cytokinesis.

scholarly journals Morphology of gametes, post-fertilization events and the effect of temperature on the embryonic development of Astyanax altiparanae (Teleostei, Characidae)

Zygote ◽  
2016 ◽  
Vol 24 (6) ◽  
pp. 795-807 ◽  
Author(s):  
Matheus Pereira dos Santos ◽  
George Shigueki Yasui ◽  
Pedro Luiz Porfírio Xavier ◽  
Nadya Soares de Macedo Adamov ◽  
Nivaldo Ferreira do Nascimento ◽  
...  
Keyword(s):  
Developmental Stages ◽  
Polar Body ◽  
Effect Of Temperature ◽  
Male And Female ◽  
Fundamental Information ◽  
Spherical Head ◽  
Typical Morphology ◽  
Female Pronucleus ◽  
Second Polar Body ◽  
First Time

SummaryThe aim of this study was to describe the morphology of gametes, post-fertilization events and subsequent temperature effects on the early developmental stages of the neotropical species Astyanax altiparanae. The sperm of this species presents a typical morphology of teleost sperm with a spherical head (diameter = 1.88 µm), midpiece (diameter = 0.75 µm) and a single flagellum (length = 18.67 µm). The extrusion of the second polar body and fusion of male and female pronucleus were reported for the first time in this species. Additionally, we observed the formation of the fertilization cone, which prevents polyspermic fertilization. Developmental stages at 22°C, 26°C and 30°C gave rise to fertilization rates at 91.12, 91.42 and 93.04% respectively. Hatching occurred at 25 hpf at 22°C, 16 hpf at 26°C and 11 hpf at 30°C and the hatching rates were 61.78%, 62.90% and 59.45%, respectively. At 22°C, the second polar body was extruded at ≈6 mpf and the male and female pronucleus fused at ≈10 mpf. This fundamental information is important for the field and opens up new possibilities in fish biotechnology, including micromanipulation and chromosome-set manipulation.

scholarly journals Roles of heterotrimeric and monomeric G proteins in sperm-induced activation of mouse eggs

Development ◽  
1994 ◽  
Vol 120 (11) ◽  
pp. 3313-3323 ◽  
Author(s):  
G.D. Moore ◽  
T. Ayabe ◽  
P.E. Visconti ◽  
R.M. Schultz ◽  
G.S. Kopf
Keyword(s):  
G Protein ◽  
G Proteins ◽  
Zona Pellucida ◽  
Polar Body ◽  
Active Form ◽  
Dominant Negative ◽  
Egg Activation ◽  
Cell Stage ◽  
Second Polar Body ◽  
Mouse Eggs

Results of several lines of experimentation suggest that sperm-induced egg activation has several features in common with G protein-coupled receptor signal transduction mechanisms. We report that microinjection of GDP beta S into metaphase II-arrested mouse eggs blocks sperm-induced egg activation. Since GDP beta S inactivates both heterotrimeric and monomeric classes of G proteins, the involvement of members of each of these families in sperm-induced egg activation was evaluated. Neither pertussis toxin treatment of eggs nor microinjection of eggs with inhibitory antibodies toward G alpha q blocked sperm-induced egg activation. Nevertheless, microinjection of phosducin, a protein that binds tightly to free G protein beta gamma subunits, specifically inhibited second polar body emission, the fertilization evoked decrease of H1 kinase activity and pronucleus formation. Microinjection of phosducin, however, did not inhibit the fertilization-induced modifications of the zona pellucida and microinjection of beta gamma t did not result in egg activation in the absence of sperm. Inactivation of the monomeric Rho family of G proteins with C3 transferase from Clostridium botulinum inhibited emission of the second polar body and cleavage to the 2-cell stage, but did not affect the modifications of the zona pellucida or pronucleus formation. Microinjection of Rasval12, which is a constitutively active form of Ras, did not result in egg activation in the absence of sperm. Moreover, microinjection of either an anti-Ras neutralizing antibody (Y13-259) or a dominant negative form of Ras (RasT) did not affect events of sperm-induced egg activation. In contrast, microinjection of RasT inhibited embryo cleavage to the 2-cell stage. These results suggest that both heterotrimeric and monomeric G proteins are involved in various aspects of sperm-induced egg activation.

Parthenogenetic development of mouse embryos in vivo

Development ◽  
1973 ◽  
Vol 30 (3) ◽  
pp. 519-545
Author(s):  
Anna Witkowska
Keyword(s):  
Polar Body ◽  
Subsequent Development ◽  
Hormonal Treatment ◽  
Inbred Strains ◽  
Early Cleavage ◽  
Parthenogenetic Development ◽  
Second Polar Body ◽  
First Time

Newly ovulated eggs from CBA-p and CBA-T6T6 inbred strains were activated in situ with an electric shock of 20–50 V. Both spontaneously ovulating females mated with vasectomized males (strain A) and those induced to ovulate and kept apart from males were used. The immediate reaction of eggs was examined 5–7 h after activation and their subsequent development was followed every day up to the 6th day (eggs examined on the 5th and 6th day were tube-locked by a ligature). In spontaneous ovulators the highest yield of activated eggs was obtained following 30 V shock (75%), with a mean for four series (20, 30, 40 and 50 V) of 46·1%. The most common reaction is extrusion of the second polar body (2 PB) and formation of one pronucleus (about 70% of activated eggs). Other types of reaction include: immediate cleavage, suppression of 2 PB with formation of two or, very rarely, one pronucleus. Thus the majority of eggs are potentially haploid. No relationship between the frequency of various types of reaction and the strength of the shock (voltage) was observed. Up to the 3rd day cleavage proceeds at a normal rate and appears to slow down during the 4th day. In spontaneously ovulating females the incidence of blastocysts among developing eggs rises from 3·4% on the 4th day to 52·5% on the 5th day; the corresponding figures for induced ovulations are 10% and 23·6%. Some embryos despite being composed of a large number of cells (40–120) do not undergo cavitation and remain morulae. On the basis of karyological examination of 60 embryos with analysable metaphase plates 60% were classified as haploid, 16·7% as diploid, 1·7% as tetraploid and 21·6% as haploid/diploid mosaics. During early cleavage binucleate blastomeres (a presumed step in regulation from n to 2n) were observed only in eggs from induced ovulations. In embryos developed from spontaneously ovulated eggs n/2n mosaics were observed for the first time on the 5th day. In general, spontaneously ovulated eggs developed better after activation (both in quantitative and qualitative terms) than those obtained following hormonal treatment.

scholarly journals Differential Expression and Functions of Cortical Myosin IIA and IIB Isotypes during Meiotic Maturation, Fertilization, and Mitosis in Mouse Oocytes and Embryos

1998 ◽  
Vol 9 (9) ◽  
pp. 2509-2525 ◽  
Author(s):  
Calvin Simerly ◽  
Grzegorz Nowak ◽  
Primal de Lanerolle ◽  
Gerald Schatten
Keyword(s):  
Differential Expression ◽  
Polar Body ◽  
Myosin Ii ◽  
Meiotic Spindle ◽  
Meiotic Maturation ◽  
Myosin Isoforms ◽  
Cleavage Furrow ◽  
Myosin Iia ◽  
Second Polar Body

To explore the role of nonmuscle myosin II isoforms during mouse gametogenesis, fertilization, and early development, localization and microinjection studies were performed using monospecific antibodies to myosin IIA and IIB isotypes. Each myosin II antibody recognizes a 205-kDa protein in oocytes, but not mature sperm. Myosin IIA and IIB demonstrate differential expression during meiotic maturation and following fertilization: only the IIA isoform detects metaphase spindles or accumulates in the mitotic cleavage furrow. In the unfertilized oocyte, both myosin isoforms are polarized in the cortex directly overlying the metaphase-arrested second meiotic spindle. Cortical polarization is altered after spindle disassembly with Colcemid: the scattered meiotic chromosomes initiate myosin IIA and microfilament assemble in the vicinity of each chromosome mass. During sperm incorporation, both myosin II isotypes concentrate in the second polar body cleavage furrow and the sperm incorporation cone. In functional experiments, the microinjection of myosin IIA antibody disrupts meiotic maturation to metaphase II arrest, probably through depletion of spindle-associated myosin IIA protein and antibody binding to chromosome surfaces. Conversely, the microinjection of myosin IIB antibody blocks microfilament-directed chromosome scattering in Colcemid-treated mature oocytes, suggesting a role in mediating chromosome–cortical actomyosin interactions. Neither myosin II antibody, alone or coinjected, blocks second polar body formation, in vitro fertilization, or cytokinesis. Finally, microinjection of a nonphosphorylatable 20-kDa regulatory myosin light chain specifically blocks sperm incorporation cone disassembly and impedes cell cycle progression, suggesting that interference with myosin II phosphorylation influences fertilization. Thus, conventional myosins break cortical symmetry in oocytes by participating in eccentric meiotic spindle positioning, sperm incorporation cone dynamics, and cytokinesis. Although murine sperm do not express myosin II, different myosin II isotypes may have distinct roles during early embryonic development.

scholarly journals Expression of BARD1 β Isoform in Selected Pediatric Tumors

Genes ◽  
2021 ◽  
Vol 12 (2) ◽  
pp. 168
Author(s):  
Anna Jasiak ◽  
Natalia Krawczyńska ◽  
Mariola Iliszko ◽  
Katarzyna Czarnota ◽  
Kamil Buczkowski ◽  
...  
Keyword(s):  
Pediatric Cancer ◽  
Genome Stability ◽  
Cell Cycle Control ◽  
Reverse Transcriptase Gene ◽  
Neoplastic Tissue ◽  
Adult Type ◽  
Qpcr Method ◽  
New Treatments ◽  
First Time ◽  
Work Supports

Currently, many new possible biomarkers and mechanisms are being searched and tested to analyse pathobiology of pediatric tumours for the development of new treatments. One such candidate molecular factor is BARD1 (BRCA1 Associated RING Domain 1)—a tumour-suppressing gene involved in cell cycle control and genome stability, engaged in several types of adult-type tumours. The data on BARD1 significance in childhood cancer is limited. This study determines the expression level of BARD1 and its isoform beta (β) in three different histogenetic groups of pediatric cancer—neuroblastic tumours, and for the first time in chosen germ cell tumours (GCT), and rhabdomyosarcoma (RMS), using the qPCR method. We found higher expression of beta isoform in tumour compared to healthy tissue with no such changes concerning BARD1 full-length. Additionally, differences in expression of BARD1 β between histological types of neuroblastic tumours were observed, with higher levels in ganglioneuroblastoma and ganglioneuroma. Furthermore, a higher expression of BARD1 β characterized yolk sac tumours (GCT type) and RMS when comparing with non-neoplastic tissue. These tumours also showed a high expression of the TERT (Telomerase Reverse Transcriptase) gene. In two RMS cases we found deep decrease of BARD1 β in post-chemotherapy samples. This work supports the oncogenicity of the beta isoform in pediatric tumours, as well as demonstrates the differences in its expression depending on the histological type of neoplasm, and the level of maturation in neuroblastic tumours.

scholarly journals Mutational analyses of fs(1)Ya, an essential, developmentally regulated, nuclear envelope protein in Drosophila.

Genetics ◽  
1995 ◽  
Vol 141 (4) ◽  
pp. 1473-1481 ◽  
Author(s):  
J Liu ◽  
K Song ◽  
M F Wolfner
Keyword(s):  
Cell Cycle ◽  
Embryonic Development ◽  
Nuclear Envelope ◽  
Envelope Protein ◽  
Chromatin Condensation ◽  
Cell Cycle Control ◽  
Nuclear Lamina ◽  
Mitotic Division ◽  
Developmentally Regulated ◽  
Egg Maturation

Abstract The fs(1)Ya protein (YA) is an essential, maternally encoded, nuclear lamina protein that is under both developmental and cell cycle control. A strong Ya mutation results in early arrest of embryos. To define the function of YA in the nuclear envelope during early embryonic development, we characterized the phenotypes of four Ya mutants alleles and determined their molecular lesions. Ya mutant embryos arrest with abnormal nuclear envelopes prior to the first mitotic division; a proportion of embryos from two leaky Ya mutants proceed beyond this but arrest after several abnormal divisions. Ya unfertilized eggs contain nuclei of different sizes and condensation states, apparently due to abnormal fusion of the meiotic products immediately after meiosis. Lamin is localized at the periphery of the uncondensed nuclei in these eggs. These results suggest that YA function is required during and after egg maturation to facilitate proper chromatin condensation, rather than to allow a lamin-containing nuclear envelope to form. Two leaky Ya alleles that partially complement have lesions at opposite ends of the YA protein, suggesting that the N- and C-termini are important for YA function and that YA might interact with itself either directly or indirectly.

Preservation of sperm within the mouse cauda epididymidis in salt or sugars at room temperature

Zygote ◽  
2010 ◽  
Vol 18 (3) ◽  
pp. 245-256 ◽  
Author(s):  
Tetsuo Ono ◽  
Eiji Mizutani ◽  
Chong Li ◽  
Teruhiko Wakayama
Keyword(s):  
Room Temperature ◽  
Polar Body ◽  
Methylation Status ◽  
Cost Effective ◽  
Oocyte Activation ◽  
Male Gametes ◽  
Effective Manner ◽  
Cell Embryo ◽  
Cauda Epididymidis ◽  
Second Polar Body

SummaryThe development of preservation techniques for male gametes at room temperature might allow us to store them in a simple and cost-effective manner. In this study, we studied the use of pure salt or sugar to preserve the whole cauda epididymidis, because it is known that food can be preserved in this way at room temperature for long periods. Mouse epididymides were placed directly in powdered salt (NaCl) or sugars (glucose or raffinose) for 1 day to 1 year at room temperature. Spermatozoa were recovered from the preserved organs after being rehydrated with medium and then isolated sperm heads were microinjected into fresh oocytes. Importantly, the oocyte activation capacity of spermatozoa was maintained after epididymal storage in NaCl for 1 year, whereas most untreated spermatozoa failed to activate oocytes within 1 month of storage. Pronuclear morphology, the rate of extrusion of a second polar body and the methylation status of histone H3 lysine 9 (H3K9me3) in those zygotes were similar to those of zygotes fertilized with fresh spermatozoa. However, the developmental ability of the zygotes decreased within 1 day of sperm storage. This effect led to nuclear fragmentation at the 2-cell embryo stage, irrespective of the storage method used. Thus, although the preserved sperm failed to allow embryo development, their oocyte activation factors were maintained by salt storage of the epididymis for up to 1 year at room temperature.

Maturation promoting factor in ascidian oocytes is regulated by different intracellular signals at meiosis I and II

Development ◽  
1996 ◽  
Vol 122 (7) ◽  
pp. 1995-2003 ◽  
Author(s):  
G.L. Russo ◽  
K. Kyozuka ◽  
L. Antonazzo ◽  
E. Tosti ◽  
B. Dale
Keyword(s):  
Kinase Activity ◽  
Phase 1 ◽  
Polar Body ◽  
Calcium Transients ◽  
Phase 2 ◽  
Phase 3 ◽  
First Polar Body ◽  
Second Polar Body ◽  
Polar Body Extrusion ◽  
Maturation Promoting Factor

Using the fluorescent dye Calcium Green-dextran, we measured intracellular Ca2+ in oocytes of the ascidian Ciona intestinalis at fertilization and during progression through meiosis. The relative fluorescence intensity increased shortly after insemination in a single transient, the activation peak, and this was followed by several smaller oscillations that lasted for approximately 5 minutes (phase 1). The first polar body was extruded after the completion of the phase 1 transients, about 9 minutes after insemination, and then the intracellular calcium level remained at baseline for a period of 5 minutes (phase 2). At 14 minutes postinsemination a second series of oscillations was initiated that lasted 11 minutes (phase 3) and terminated at the time of second polar body extrusion. Phases 1 and 3 were inhibited by preloading oocytes with 5 mM heparin. Simultaneous measurements of membrane currents, in the whole-cell clamp configuration, showed that the 1–2 nA inward fertilization current correlated temporally with the activation peak, while a series of smaller oscillations of 0.1-0.3 nA amplitude were generated at the time of the phase 3 oscillations. Biochemical characterization of Maturation Promoting Factor (MPF) in ascidian oocytes led to the identification of a Cdc2-like kinase activity. Using p13suc1-sepharose as a reagent to precipitate the MPF complex, a 67 kDa (67 × 10(3) Mr) protein was identified as cyclin B. Histone H1 kinase activity was high at metaphase I and decreased within 5 minutes of insemination reaching a minimum level during phase 2, corresponding to telophase I. During phase 3, H1 kinase activity increased and then decayed again during telophase II. Oocytes preloaded with BAPTA and subsequently inseminated did not generate any calcium transients, nonetheless H1 kinase activity decreased 5 minutes after insemination, as in the controls, and remained low for at least 30 minutes. Injection of BAPTA during phase 2 suppressed the phase 3 calcium transients, and inhibited both the increase in H1 kinase activity normally encountered at metaphase II and second polar body extrusion.

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