scholarly journals Somatic cell nuclear transfer in non-enucleated goldfish oocytes: understanding DNA fate during meiosis resumption and first cellular division

2019 ◽  
Author(s):  
Charlène Rouillon ◽  
Alexandra Depincé ◽  
Nathalie Chênais ◽  
Pierre-Yves Le Bail ◽  
Catherine Labbé
Keyword(s):  
Cell Cycle ◽  
Mitotic Spindle ◽  
Nuclear Transfer ◽  
Polar Body ◽  
Somatic Nucleus ◽  
Cellular Division ◽  
Dna Labeling ◽  
Cellular Events ◽  
Polar Body Extrusion ◽  
Fish Oocyte

AbstractNuclear transfer consists in injecting a somatic nucleus carrying valuable genetic information into a recipient oocyte to sire a diploid offspring who bears the genome of interest. It requires that the oocyte (maternal) DNA is removed. In fish, because enucleation is difficult to achieve, non-enucleated oocytes are often used and disappearance of the maternal DNA was reported in some clones. The present work explore which cellular events explain spontaneous erasure of maternal DNA, as mastering this phenomenon would circumvent the painstaking procedure of fish oocyte enucleation. The fate of the somatic and maternal DNA during meiosis resumption and first cell cycle was studied using DNA labeling and immunofluorescence in goldfish clones. Maternal DNA was always found as an intact metaphase within the oocyte, and polar body extrusion was minimally affected after meiosis resumption. During the first cell cycle, only 40 % of the clones displayed symmetric cleavage, and these symmetric clones contributed to 80 % of those surviving at hatching. Maternal DNA was often fragmented and located under the cleavage furrow. The somatic DNA was organized either into a normal mitotic spindle or abnormal multinuclear spindle. Scenarios matching the DNA behavior and the embryo fate are proposed.

scholarly journals CLONAGEM POR TRANSFERÊNCIA NUCLEAR EM BUBALINOS

2017 ◽  
Vol 13 (Especial 2) ◽  
pp. 110-117
Author(s):  
Aline Sousa Camargos ◽  
Ariane Dantas
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Nuclear Transfer ◽  
Donor Cell ◽  
Somatic Nucleus ◽  
Number Of Species ◽  
Stage Of Development ◽  
Cellular Development

The success of core transfer (CT) depends on the origin of the donor cell, on the stage of development of the recipient cytoplast and on the synchronization between the cell cycle of the donor and recipient cells. The somatic nucleus must be reprogrammed after CT, thus restoring the totipotent state, and then resuming cellular development. However, it is noted that the efficiency of CT is still low, especially with a deficiency of the overall gene expression of the cloned embryo. However, the number of species of cloned mammals has been increasing in the last years, being this technique an important tool that does not aid in the effectiveness of buffalo reproduction. Thus, this review focuses on the description of the main processes pertinent to this process, as well as to analyze as future implications, as well as some factors that affect the success of nuclear transfer.

scholarly journals 51DEVELOPMENT OF BOVINE CLONES CONSTRUCTED WITH CYTOPLASM ORIGINATING FROM OOCYTES CULTURED IN RETINOL

2004 ◽  
Vol 16 (2) ◽  
pp. 147
Author(s):  
J.L. Lawrence ◽  
F.N. Schrick ◽  
J.D. Godkin ◽  
J.L. Edwards
Keyword(s):  
Granulosa Cells ◽  
Nuclear Transfer ◽  
Block Design ◽  
Polar Body ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Somatic Nucleus ◽  
Ovarian Granulosa Cells ◽  
Electrical Pulses

Incomplete reprogramming of a somatic nucleus by the oocyte cytoplasm may contribute in large part to inefficiencies of cloning procedures using somatic cell nuclear transfer. Predominant use of cytoplasts derived from in vitro-matured oocytes may further exacerbate problems. Addition of all-trans-retinol (Livingston TL et al. 2002 Biol. Reprod. 66, 104–105 abst) or 9-cis-retinoic acid (Duque P et al. 2002 Human Reprod. 17, 2706–2714) to culture medium improved developmental competence of oocytes to blastocyst stage. The objective of this study was to evaluate effects of retinol for improving developmental competence of cloned embryos constructed with retinol-treated cytoplasts. After removal from 3–8mm follicles, COC were matured in the presence of 0 (n=1005; diluent only) or 5μM all trans-retinol (n=1017). Beginning approximately 18h after placement into culture, oocytes with extruded polar bodies were enucleated of maternal chromatin. Ovarian/granulosa cells were aspirated from adult Jersey cows (n=2) using an ultrasound-guided transvaginal probe. Primary cell lines were established and, before nuclear transfer, cultured in the presence of 0.5% serum (5–8 days; passage =3). Ovarian/granulosa cells were fused with cytoplasts originating from control or retinol-treated oocytes using two electrical pulses of 2.2kVcm−1 for 20μs between 24–27h post-maturation (hpm). Cloned embryos were activated between 26.5 and 30hpm and then cultured in an atmosphere of 7% O2 and 5.5% CO2 in KSOMaa+BSA. Development of cloned embryos to morula and blastocyst stages was assessed on Days 6–7 post-activation. In 5 replicates, compact morulae and blastocysts were transferred to synchronous recipients. Establishment of pregnancy was confirmed 28 days post-estrus by presence of an embryonic heartbeat using ultrasound. With the exception of pregnancy, data were analyzed as a randomized block design using mixed models of SAS (2000) after testing for normality. Proportion of oocytes recovered after denuding (94.5 and 88.9%; SEM=2.2), those that had visibly lysed (6.4 and 7.3%; SEM=1.5), and extruded a polar body by 20hpm (61.7 and 62.5%; SEM=7.0) was similar for control and retinol-treated oocytes, respectively. In addition, ability of ovarian/granulosa cells to fuse with control or retinol-treated cytoplasts was similar (80.6 and 73.1%; SEM=4.5). Lysis of cytoplasts after electrical pulses (8.2 v. 13.6%; SEM=3.3) and activation (11.6 v. 5.0%; SEM=2.9) did not differ for control and retinol-treated cytoplasts. Development to morula and blastocyst stages was lower in cloned embryos constructed with retinol-treated cytoplasts (Table). However, ability of morulae and blastocysts to establish a pregnancy was comparable. One clone from each treatment developed to term and was born alive. Culture of oocytes in medium containing retinol during maturation did not improve developmental competence of clones.

290. Calmodulin-dependent protein kinase II, and not protein kinase C, transduces the Ca2+ signal at fertilization

2005 ◽  
Vol 17 (9) ◽  
pp. 122
Author(s):  
K. T. Jones ◽  
M. Levasseur ◽  
H. Chang ◽  
S. Madgwick
Keyword(s):  
Cell Cycle ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Polar Body ◽  
Dependent Protein Kinase ◽  
Kinase C ◽  
Dependent Protein ◽  
Polar Body Extrusion ◽  
Mouse Eggs ◽  
Constitutively Active

Mouse eggs arrest at metaphase II following ovulation and are only triggered to complete meiosis when fertilized. Sperm break the cell cycle arrest by a long-lasting series of Ca2+ spikes that lead to an activation of the anaphase-promoting complex. The signal transduction pathway is not fully resolved but both protein kinase C (PKC) and calmodulin-dependent protein kinase II (CamKII) activities increase at fertilization and previous pharmacological studies have implicated both in cell cycle resumption. Here we used a combination of pharmacological inhibitors and constitutively-active cRNA constructs of PKCα and CamKII microinjected into mouse eggs, to show that it is CamKII and not PKC that is the sufficient trigger for cell cycle resumption from metaphase II arrest. Constitutively active PKC constructs had no effect on meiotic resumption but caused an immediate and persistent elevation in intracellular Ca2+ when store-operated Ca2+ entry was stimulated. With respect to meiotic resumption, the effects of constitutively-active CamKII on eggs were the same as sperm. Eggs underwent second polar body extrusion and pronucleus formation with normal timings; while both securin and cyclin B1 destruction, visualised by coupling to fluorescent protein tags, were complete by the time of polar body extrusion. Induction of a spindle checkpoint by overexpression of Mad2 or by spindle poisons blocked CamKII-induced meiotic resumption but the Ca2+ chelator BAPTA did not. Furthermore direct measurement of Ca2+ levels showed that CamKII did not induce exit from metaphase II arrest by raising Ca2+. Therefore we conclude that PKCs may play an important role in maintaining Ca2+ spiking at fertilization by promoting store-operated Ca2+ entry, while CamKII transduces cell cycle resumption, and lies downstream of sperm-induced Ca2+ release but upstream of a spindle checkpoint. These data, combined with the knowledge that CamKII activity increase at fertilization, suggest that mouse eggs undergo cell cycle resumption through stimulation of CamKII.

scholarly journals 011.Waking up the egg. How the sperm regulates exit out of the meiotic cell cycle

2004 ◽  
Vol 16 (9) ◽  
pp. 11
Author(s):  
K. T. Jones
Keyword(s):  
Cell Cycle ◽  
Polar Body ◽  
Cyclin B1 ◽  
26S Proteasome ◽  
Calcium Signal ◽  
Calcium Spikes ◽  
M Phase ◽  
Second Polar Body ◽  
Polar Body Extrusion ◽  
Mammalian Eggs

A series of calcium spikes are induced in the mammalian egg cytoplasm at fertilisation. These calcium spikes, which last for several hours, are the necessary and sufficient signal that stimulates the egg to escape from arrest at metaphase of the second meiotic division. Metaphase arrest is achieved by preventing the destruction of cyclin B1, the regulatory component of Maturation (M-Phase) Promoting Factor, and securin, which prevents segregation of sister chromatids. Both these proteins are destroyed by tagging with ubiquitin, using an E3 ligase the Anaphase-Promoting Complex (APC). Ubiquitination tags them for proteolysis by the 26S proteasome. Work from my lab has demonstrated that the sperm calcium signal works through activating the APC, not the 26S proteasome. Although we do not know which APC component is affected by calcium, this activation appears specific to a metaphase-arrested cell cycle state. More recently we have found that the APC is differently regulated at specific points during exit from meiosis II. Before extrusion of the second polar body it is the APC activator cdc20 that regulates APC activity. However, following extrusion of the second polar body cdh1 appears the major regulator. It is probable, therefore, that the calcium spiking affects the activity of both APCcdc20 and APCcdh1. This swap in APC activator at the time of second polar body extrusion has not been reported in eggs of other species, in fact non-mammalian eggs all lack cdh1. Since APCcdc20 and APCcdh1 have different substrate specificities, the function of APCcdh1 in mammalian eggs warrants further investigation.

scholarly journals Ral GTPase is essential for actin dynamics and Golgi apparatus distribution in mouse oocyte maturation

Cell Division ◽  
2021 ◽  
Vol 16 (1) ◽  
Author(s):  
Ming-Hong Sun ◽  
Lin-Lin Hu ◽  
Chao-Ying Zhao ◽  
Xiang Lu ◽  
Yan-Ping Ren ◽  
...  
Keyword(s):  
Golgi Apparatus ◽  
Oocyte Maturation ◽  
Polar Body ◽  
Mouse Oocyte ◽  
Actin Dynamics ◽  
Mouse Oocytes ◽  
Oocyte Meiosis ◽  
Ral Gtpase ◽  
Polar Body Extrusion ◽  
Cytoskeleton Dynamics

Abstract Background Ral family is a member of Ras-like GTPase superfamily, which includes RalA and RalB. RalA/B play important roles in many cell biological functions, including cytoskeleton dynamics, cell division, membrane transport, gene expression and signal transduction. However, whether RalA/B involve into the mammalian oocyte meiosis is still unclear. This study aimed to explore the roles of RalA/B during mouse oocyte maturation. Results Our results showed that RalA/B expressed at all stages of oocyte maturation, and they were enriched at the spindle periphery area after meiosis resumption. The injection of RalA/B siRNAs into the oocytes significantly disturbed the polar body extrusion, indicating the essential roles of RalA/B for oocyte maturation. We observed that in the RalA/B knockdown oocytes the actin filament fluorescence intensity was significantly increased at the both cortex and cytoplasm, and the chromosomes were failed to locate near the cortex, indicating that RalA/B regulate actin dynamics for spindle migration in mouse oocytes. Moreover, we also found that the Golgi apparatus distribution at the spindle periphery was disturbed after RalA/B depletion. Conclusions In summary, our results indicated that RalA/B affect actin dynamics for chromosome positioning and Golgi apparatus distribution in mouse oocytes.

FASCIN regulates actin assembly for spindle movement and polar body extrusion in mouse oocyte meiosis

2021 ◽  
Author(s):  
Lin‐Lin Hu ◽  
Meng‐Hao Pan ◽  
Feng‐Lian Yang ◽  
Zi‐Ao Zong ◽  
Feng Tang ◽  
...  
Keyword(s):  
Polar Body ◽  
Mouse Oocyte ◽  
Actin Assembly ◽  
Oocyte Meiosis ◽  
Polar Body Extrusion

scholarly journals Definition of conditions that enable antigen-specific activation of the majority of isolated trinitrophenol-binding B cells.

1982 ◽  
Vol 156 (6) ◽  
pp. 1635-1649 ◽  
Author(s):  
J C Cambier ◽  
J G Monroe ◽  
M J Neale
Keyword(s):  
Cell Cycle ◽  
T Cell ◽  
Interleukin 1 ◽  
Isolated Cells ◽  
Cellular Events ◽  
Accessory Cells ◽  
B Cell Growth Factor ◽  
Human Gamma Globulin ◽  
Definition Of ◽  
T Cell Help

In an effort to further elucidate the early cellular events in generation of antibody responses, we have determined the requirements for antigen-specific initiation of the G0 to G1 transition by isolated trinitrophenol (TNP) -binding B lymphocytes. TNP-binding cells were isolated from normal B6D2F1 splenocyte populations using hapten affinity fractionation on disulfide-bonded TNP-gelatin-coated plates. Populations prepared in this way are greater than or equal to 96% immunoglobulin positive and 70-95% antigen binding. Isolated cells were cultured for 48 h in the presence of a variety of TNP conjugates including TNP-Brucella abortus (Ba), TNP-Ficoll, TNP-sheep erythrocytes (SRBC), TNP-human gamma globulin (HGG), or TNP-ovalbumin (OVA) before being harvested and subjected to acridine orange cell cycle analysis. As many as 80% of cells were in cycle by 48 h in response to TNP-Ba, a thymus-independent (TI1 antigen. A smaller proportion (congruent to 40%) were in cycle in response to TNP-Ficoll, a TI2 antigen. Significant activation was not detected in cultures challenged with the thymus-dependent immunogens TNP-SRBC, TNP-HGG, and TNP-OVA. Addition of interleukin 1 (IL-1), IL-2, B cell growth factor, and/or T cell-replacing factor to cultures did not facilitate responses to these immunogens, suggesting a requirement for antigen-specific T cell help for entry into cell cycle induced by thymus dependent antigens. Activation by TNP-Ba was antigen specific and independent of accessory cells, occurring with equal efficiency in bulk and single-cell cultures. Activation by TNP-Ba was inhibitable by anti-Fab and anti-mu antibodies, but not by anti-delta antibodies. Results indicate that activation of TNP-binding cells to enter cell cycle by TNP-Ba is independent of accessory cells and requires interaction of antigen with cell surface IgM. Exposure to thymus-dependent TNP-immunogens plus nonspecific helper factors is insufficient to cause entry of TNP-binding cells into cycle.

scholarly journals Cell cycle-dependent effects on deoxyribonucleotide and DNA labeling by nucleoside precursors in mammalian cells.

1987 ◽  
Vol 7 (1) ◽  
pp. 532-534 ◽  
Author(s):  
J M Leeds ◽  
C K Mathews
Keyword(s):  
Cell Cycle ◽  
Mammalian Cells ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
G1 Phase ◽  
Ovary Cells ◽  
Dna Labeling ◽  
Metabolic Pool ◽  
Specific Activities ◽  
Cycle Dependent

dCTP pools equilibrated to equivalent specific activities in Chinese hamster ovary cells or in nuclei after incubation of cells with radiolabeled nucleosides, indicating that dCTP in nuclei does not constitute a distinct metabolic pool. In the G1 phase, [5-3H]deoxycytidine labeled dCTP to unexpectedly high specific activities. This may explain reports of replication-excluded DNA precursor pools.

Effects of cytochalasin B on meiosis and development of fertilized and activated eggs of Sabellaria alveolata L. (Polychaete Annelid)

Development ◽  
1974 ◽  
Vol 31 (1) ◽  
pp. 61-74
Author(s):  
G. Peaucellier ◽  
P. Guerrier ◽  
J. Bergerard
Keyword(s):  
Cytochalasin B ◽  
Polar Body ◽  
Polar Lobe ◽  
Developmental Abnormalities ◽  
Drug Concentrations ◽  
Internal Mechanism ◽  
Polar Body Extrusion

1. Unfertilized, fertilized and activated eggs of Sabellaria alveolata were submitted to cytochalasin B concentrations ranging from 0·1 to 20 μg/ml. Their behaviour was studied either in vivo or in acetocarmine squash preparations. 2. Polar body extrusion, cytokinesis and polar lobe formation are completely inhibited by cytochalasin B concentrations as low as 0·3–0·5 μg/ml. 3. Caryotype determinations demonstrate that chromosomal meiotic and mitotic processes are not affected by the drug. Thus, polyploid embryos usually developed from fertilized eggs whilst they did not from activated ones. This is related to the contrasting behaviour of meiotic and cleavage centres. While the latter duplicates at each cycle, the former cannot replicate at the end of meiosis. This leads to an abortive monastral stage even if inhibition of polar body extrusion has provided the egg with two or four centres. These observations suggest the existence of an internal mechanism regulating the number of effective centrioles at the end of meiosis. They demonstrate also that the main cause of developmental failure in activated eggs cannot be related to ploidy. 4. Eggs treated throughout meiosis with moderate drug concentrations developed into swimming larvae. However, frequent developmental abnormalities affecting lobe dependent structures were obtained even if polar lobe formation was unimpaired. This suggests either that cytochalasin B has irreversibly affected some decisive cortical element or that previously described activating processes, which begin with polar lobe formation, are actually exerted on specific materials segregated during meiosis.

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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