scholarly journals Dynamic changes of the Golgi apparatus during bovine in vitro oocyte maturation

Reproduction ◽  
2012 ◽  
Vol 143 (4) ◽  
pp. 439-447 ◽  
Author(s):  
S E Racedo ◽  
V Y Rawe ◽  
H Niemann
Keyword(s):  
Golgi Apparatus ◽  
Germinal Vesicle ◽  
Male Gamete ◽  
Active Role ◽  
Cytoplasmic Dynein ◽  
Dynamic Changes ◽  
Kinesin Eg5 ◽  
Specific Inhibitors

For successful fertilization by the male gamete, oocyte cytoplasmic organelles such as the Golgi apparatus have to undergo specific changes: the entire process is known as cytoplasmic maturation. The goal of this study was to unravel the dynamics of the Golgi apparatus in bovine oocytes at critical stages ofin vitromaturation, i.e. germinal vesicle (GV), GV breakdown (GVBD), metaphase I (MI) and metaphase II, and to investigate the role of various molecules critically involved therein. The cytoplasmic distribution of proteins was assessed by immunocytochemistry and laser confocal microscopy. We applied specific inhibitors, including nocodazole to unravel the functional role of the microtubular elements; sodium orthovanadate, which primarily inhibits cytoplasmic dynein ATPase activity; monastrol which inhibits the kinesin EG5; and roscovitine to inhibit the kinase cyclin-dependent kinase 2A (CDC2A). Prior to GVBD, the Golgi apparatus was translocated from the centre of the cytoplasm to the cortical area in the periphery, where it underwent fragmentation. A second translocation was observed between GVBD and MI stages, when the Golgi apparatus was moved from the cortex to the centre of the cytoplasm. Incubation with the specific inhibitors revealed that microtubules played an active role in the final localization at GVBD, while CDC2A was essential for Golgi fragmentation at GVBD stage. This partitioning was a precondition for the second movement. In conclusion, for the first time we show basic mechanisms critically involved in the regulation of the dynamic changes of Golgi apparatus during meiosis of the bovine oocyte.

254 DYNAMICS OF GOLGI APPARATUS DURING BOVINE OOCYTE IN VITRO MATURATION: EFFECTS OF INHIBITORS ON CDC2A AND CYTOPLASMIC-DYNEIN ATPase ACTIVITY

2009 ◽  
Vol 21 (1) ◽  
pp. 225
Author(s):  
S. E. Racedo ◽  
V. Y. Rawe ◽  
H. Niemann
Keyword(s):  
Golgi Apparatus ◽  
Atpase Activity ◽  
Oocyte Maturation ◽  
In Vitro Maturation ◽  
Germinal Vesicle ◽  
Cytoplasmic Dynein ◽  
Bovine Oocyte ◽  
Germinal Vesicle Breakdown

The process of maturation encompasses a complex series of molecular and structural events. Completion of the nuclear changes to produce a metaphase II (MII) oocyte does not reflect the molecular and structural maturity of an oocyte, which is sometimes termed cytoplasmic maturation. The Golgi apparatus phosphorylates, fragments, and changes the localization during oocyte maturation. GM130 and phospho-GM130 are used as markers for the Golgi apparatus and phosphorylated Golgi apparatus, respectively. The goal of this study was to analyze the dynamics of the Golgi apparatus and its association with microtubules in bovine oocytes at different stages of in vitro maturation [IVM; i.e. germinal vesicle (GV), germinal vesicle breakdown (GVBD), metaphase I (MI), and MII]. The roles of CDC2A kinase (also known as p34cdc2) and cytoplasmic-dynein ATPase on Golgi dynamics were studied by using specific inhibitors. The distribution of the markers was assessed by immunocytochemistry and laser confocal microscopy. To unravel the role of CDC2A and cytoplasmic dynein ATPase on the dynamics of the Golgi apparatus, the inhibitors roscovitine (ROS) and sodium-orthovanadate (SOV), respectively, were used. In the first experiment, the nuclear maturation rate was checked in the presence of the inhibitors at different times and for different incubation times to explore whether oocytes were able to reach the MII stage. At the GV and GVBD stages, the Golgi apparatus is observed as fragments named mini-Golgies and at the MI and MII stages as punctate foci throughout the cytoplasm. Our results showed 2 well-defined movements of the Golgi apparatus toward opposite directions, depending on the maturation stage. The first movement was observed between 5 and 9 h of IVM (i.e. the GVBD stage), when the Golgi apparatus relocalized from the ooplasm to the periphery. The second movement was observed between 9 and 15 h of IVM (i.e. the MI stage), when the Golgi apparatus moved from the cortex to throughout the cytoplasm and remained there up to the MII stage. The use of inhibitors on CDC2A and cytoplasmic-dynein ATPase at selected time points revealed that CDC2A played a crucial role on the distribution of this organelle during the first movement, whereas the final localization at the GVBD stage was dependent on cytoplasmic-dynein transport. The second movement of the Golgi apparatus was disturbed by the SOV treatment, but not by the use of ROS, suggesting a role of cytoplasmic-dynein-dependent transport during the distribution and organization of the punctate foci at the MI stage. The phosphorylation status of Golgi was not affected at the different incubation times with inhibitors, except in those oocytes incubated with ROS for 24 h, suggesting a role of CDC2A. In conclusion, we describe the involvement of CDC2A during the first movement of the Golgi apparatus and the importance of cytoplasmic-dynein ATPase activity in the first and second relocalization of Golgi during bovine oocyte maturation. DAAD.

scholarly journals Requirement of mosXe protein kinase for meiotic maturation of Xenopus oocytes induced by a cdc2 mutant lacking regulatory phosphorylation sites.

1992 ◽  
Vol 12 (7) ◽  
pp. 3192-3203 ◽  
Author(s):  
K M Pickham ◽  
A N Meyer ◽  
J Li ◽  
D J Donoghue
Keyword(s):  
Protein Kinase ◽  
Oocyte Maturation ◽  
Xenopus Oocytes ◽  
Germinal Vesicle ◽  
Cyclin B1 ◽  
Phosphorylation Sites ◽  
Germinal Vesicle Breakdown ◽  
Regulatory Phosphorylation

The p34cdc2 protein kinase is a component of maturation-promoting factor, the master regulator of the cell cycle in all eukaryotes. The activity of p34cdc2 is itself tightly regulated by phosphorylation and dephosphorylation. Predicted regulatory phosphorylation sites of Xenopus p34cdc2 were mutated in vitro, and in vitro-transcribed RNAs were injected into Xenopus oocytes. The cdc2 single mutants Thr-14----Ala and Tyr-15----Phe did not induce germinal vesicle breakdown (BVBD) upon microinjection into oocytes. In contrast, the cdc2 double mutant Ala-14/Phe-15 did induce GVBD. Both the Ala-14 and Ala-14/Phe-15p34cdc2 mutants were shown to coimmunoprecipitate cyclin B1 and to phosphorylate histone H1 in immune complex kinase assays. Microinjection of antisense oligonucleotides to c-mosXe was used to demonstrate the role of mos protein synthesis in the induction of GVBD by the Ala-14/Phe-15 cdc2 mutant. Thr-161 was also mutated. p34cdc2 single mutants Ala-161 and Glu-161 and triple mutants Ala-14/Phe-15/Ala-161 and Ala-14/Phe-15/Glu-161 failed to induce GVBD in oocytes and showed a decreased binding to cyclin B1 in coimmunoprecipitations. Each of the cdc2 mutants was also assayed by coinjection with cyclin B1 or c-mosXe RNA into oocytes. Several of the cdc2 mutants were found to affect the kinetics of cyclin B1 and/or mos-induced GVBD upon coinjection, although none affected the rate of progesterone-induced maturation. We demonstrate here the significance of Thr-14, Tyr-15, and Thr-161 of p34cdc2 in Xenopus oocyte maturation. In addition, these results suggest a regulatory role for mosXe in induction of oocyte maturation by the cdc2 mutant Ala-14/Phe-15.

Neural induction and the structure of the target cell surface

Development ◽  
1982 ◽  
Vol 70 (1) ◽  
pp. 171-187
Author(s):  
A. M. Duprat ◽  
L. Gualandris ◽  
P. Rouge
Keyword(s):  
Cell Surface ◽  
Structural Integrity ◽  
Neural Induction ◽  
Active Role ◽  
Target Cells ◽  
Similar Treatment ◽  
Structural Modifications ◽  
Pleurodeles Waltl

Lectins (SBA and PSA) were used to provoke crowding and structural modifications of the presumptive ectoderm cell surface in order to investigate the role of the membrane organization of the competent target cells in neural induction. Are specific characteristics of the cell surface essential for this phenomenon to occur? From amphibian gastrulae, it is possible to obtain neural induction in vitro by association of presumptive ectoderm (target cells) with chordamesoderm (inductor tissue): 4 h of contact is sufficient in Pleurodeles waltl for transmission of the inductive signal. Very quickly, the treatment of the normal ectoderm by lectins (SBA-FITC or PSA-FITC) provoked surface modifications. Lectin-treatment (50 µg ml1−, 30 min) of presumptive ectoderm did not result in any neural induction. Lectin-treatment (50 µg ml1−, 30 min) of presumptive ectoderm previous to its association with the natural inductor for 4 h, disturbed the phenomenon: no induction. Similar treatment followed by association with the inductor for 24 h: induction. Treatment of SBA or PSA with their respective hapten inhibitors prior to addition to ectodermal cells completely blocked the suppressive effects on induction. The structural integrity of the membrane of competent target cells is necessary for neural induction to occur. The cell membrane could thus play, directly or indirectly, an active role in the specificity of this process

scholarly journals Cytoplasmic Dynein Nucleates Microtubules to Organize Them into Radial Arrays In Vivo

2004 ◽  
Vol 15 (6) ◽  
pp. 2742-2749 ◽  
Author(s):  
Viacheslav Malikov ◽  
Anna Kashina ◽  
Vladimir Rodionov
Keyword(s):  
Cytoplasmic Dynein ◽  
Exact Function ◽  
Necessary And Sufficient ◽  
Stimulation Of ◽  
In Cells

Numerous evidence demonstrates that dynein is crucial for organization of microtubules (MTs) into radial arrays, but its exact function in this process is unclear. Here, we studied the role of cytoplasmic dynein in MT radial array formation in the absence of the centrosome. We found that dynein is a potent MT nucleator in vitro and that stimulation of dynein activity in cytoplasmic fragments of melanophores induces nucleation-dependent formation of MT radial array in the absence of the centrosome. This new property of dynein, in combination with its known role as an MT motor that is essential for MT array organization in the absence and presence of the centrosome, makes it a unique molecule whose activity is necessary and sufficient for the formation and maintenance of MT radial arrays in cells.

Effect of enucleation on protein synthesis during maturation of bovine oocytes in vitro

1997 ◽  
Vol 9 (6) ◽  
pp. 603 ◽  
Author(s):  
J. C. Bell ◽  
L. C. Smith ◽  
R. Rumpf ◽  
A. K. Goff
Keyword(s):  
Protein Synthesis ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Germinal Vesicle ◽  
Uv Exposure ◽  
One Dimensional ◽  
Repair Mechanisms ◽  
Bovine Oocytes

The role of the nucleus in protein synthesis reprogramming during oocyte maturation was examined in immature or mature bovine oocytes, enucleated at the germinal vesicle (GV) stage or the metaphase II (MII) stage. Cumulusoocyte complexes (COCs) were denuded before or after maturationin vitro. Denuded oocytes were (i) enucleated at the GV or MII stage (after DNA staining and ultraviolet (UV) exposure), (ii) stained and exposed to UV but not enucleated, or (iii) used as controls. After treatment, oocytes were labelled for 4 h with35S-methionine or were matured for 24 h before labelling. GV- or MII- karyoplasts and small portions of cytoplasm (cytoplasts), removed during enucleation, were also labelled. Labelled oocytes, karyoplasts or cytoplasts were prepared for one-dimensional polyacrylamide gel electrophoresis. Incorporation of labelled methionine into oocyte protein was measured. Enucleation did not affect protein synthesis reprogramming, but incorporation of 35S-methionine in immature UV-stained oocytes was high-possibly due to nuclear repair mechanisms. Protein proles of GV- and MII- karyoplasts differed from those of immature and mature oocytes. In conclusion, normal protein synthesis reprogramming in the cytoplasm can occur in the absence of the nucleus, and specic proteins are synthesized in the nuclear region.

268 ROLE OF ATPase MOTOR CYTOPLASMIC DYNEIN AND PRIMARY CILIA IN TESTICULAR MORPHOGENESIS

2015 ◽  
Vol 27 (1) ◽  
pp. 223
Author(s):  
C. Dores ◽  
I. Dobrinski
Keyword(s):  
Primary Cilia ◽  
Somatic Cells ◽  
Treated Group ◽  
Cytoplasmic Dynein ◽  
Control Group ◽  
Tubule Formation ◽  
Serum Free ◽  
Free Media

In vertebrates, the primary cilium is a nearly ubiquitous organelle present in somatic cells, but little is known about its function in the male gonad. We investigated the role of primary cilia in testis cells using in vitro formation of seminiferous tubules and in vitro culture of testicular somatic cells by inhibiting the primary cilium with CiliobrevinD, a cell-permeable, reversible chemical modulator that inhibits the major component of the organelle: ATPase motor cytoplasmic dynein. We analysed in vitro cultures for the presence of primary cilia and the activation of hedgehog signalling through translocation of Gli2 to the nuclei; in vitro tubule formation was evaluated by length and width of tubules formed. Methods: testicular cells were harvested from neonatal pigs by 2-step enzymatic digestion. Cells (50 × 106 mL–1) were plated on 100 mm Petri dishes in 15 mL of DMEM + 5% FBS + 50 U of penicillin and incubated at 37°C in 5% CO2 in air overnight, cells remaining in suspension and those slightly attached were removed and the somatic cells attached were trypsinized to obtain a single cell suspension, and then submitted to two different protocols: in vitro culture (A) or in vitro tubule formation (B), n = 5 replicates each. For A, somatic cells were replated on coverslips in 24-well plates and cultured in serum free media for 48 h, then for the treated group, 10 mM of CiliobrevinD was added for 24 h, attached cells from control and treated groups were fixed in 4% PFA and characterised by immunocytochemistry for ARL13B, Vimentin, and Gli2. For B: 1 × 106 cells were added to 24-well plates coated with 1 : 1 diluted Matrigel, the control group was kept in serum free media and to the treated group was added 20 mM CiliobrevinD at Day 0. Results: A) primary cilia were present in 89.3 ± 2.3% of cells cultured in serum-free media for the control group and Gli2 was located in the nuclei of 90.2 ± 1.2% of cells; in the CiliobrevinD-treated group the percentage of primary cilia decreased (P < 0.05) to 3.1 ± 2.5% and nuclear Gli2 to 3.9 ± 0.7; B) tubules formed in the control group were significantly longer and wider than the ones formed when CiliobrevinD was added (9.91 ± 0.35 v. 5.540 ± 1.08 mm and 339.8 ± 55.78 v. 127.2 ± 11.9 µm, respectively, P < 0.05 by Student's t-test). In conclusion, the inhibition of ATPase motor cytoplasmic dynein perturbs formation of primary cilia in testicular somatic cells, blocks Hedgehog signalling, and impairs in vitro tubule formation. Therefore, primary cilia on testicular somatic cells appear to be essential for testicular morphogenesis.Research was supported by 5 R01 OD016575-13.

Role of germinal vesicle on protein synthesis in rat oocyte during in vitro maturation

1996 ◽  
Vol 43 (2) ◽  
pp. 228-235 ◽  
Author(s):  
Li Meng ◽  
Jean Rutledge ◽  
Ying Zhu ◽  
Gerald M. Kidder ◽  
Firouz Khamsi ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
In Vitro Maturation ◽  
Germinal Vesicle

scholarly journals Antibodies Induced by Lipoarabinomannan in Bovines: Characterization and Effects on the Interaction betweenMycobacterium aviumSubsp.paratuberculosisand MacrophagesIn Vitro

2011 ◽  
Vol 2011 ◽  
pp. 1-8 ◽  
Author(s):  
Ana Jolly ◽  
Silvia Beatriz Colavecchia ◽  
Bárbara Fernández ◽  
Eloy Fernández ◽  
Silvia Leonor Mundo
Keyword(s):  
Immune Response ◽  
Mycobacterium Avium ◽  
Active Role ◽  
Macrophage Cell Line ◽  
Macrophage Cell ◽  
Specific Antibodies ◽  
Humoral Immune ◽  
Bovine Macrophage

Lipoarabinomannan (LAM) is a major glycolipidic antigen on the mycobacterial envelope. The aim of this study was to characterize the humoral immune response induced by immunization with a LAM extract in bovines and to evaluate the role of the generated antibodies in thein vitroinfection of macrophages withMycobacterium aviumsubsp.paratuberculosis(MAP). Sera from fourteen calves immunized with LAM extract or PBS emulsified in Freund's Incomplete Adjuvant and from five paratuberculosis-infected bovines were studied. LAM-immunized calves developed specific antibodies with IgG1 as the predominant isotype. Serum immunoglobulins were isolated and their effect was examined in MAP ingestion and viability assays using a bovine macrophage cell line. Our results show that the antibodies generated by LAM immunization significantly increase MAP ingestion and reduce its intracellular viability, suggesting an active role in this model.

scholarly journals Dynein 1 supports spermatid transport and spermiation during spermatogenesis in the rat testis

2018 ◽  
Vol 315 (5) ◽  
pp. E924-E948 ◽  
Author(s):  
Qing Wen ◽  
Elizabeth I. Tang ◽  
Wing-yee Lui ◽  
Will M. Lee ◽  
Chris K. C. Wong ◽  
...  
Keyword(s):  
Germ Cell ◽  
Heavy Chain ◽  
Sertoli Cells ◽  
Cytoplasmic Dynein ◽  
Seminiferous Epithelium ◽  
Organelle Transport ◽  
Cellular Events

In the mammalian testis, spermatogenesis is dependent on the microtubule (MT)-specific motor proteins, such as dynein 1, that serve as the engine to support germ cell and organelle transport across the seminiferous epithelium at different stages of the epithelial cycle. Yet the underlying molecular mechanism(s) that support this series of cellular events remain unknown. Herein, we used RNAi to knockdown cytoplasmic dynein 1 heavy chain (Dync1h1) and an inhibitor ciliobrevin D to inactivate dynein in Sertoli cells in vitro and the testis in vivo, thereby probing the role of dynein 1 in spermatogenesis. Both treatments were shown to extensively induce disruption of MT organization across Sertoli cells in vitro and the testis in vivo. These changes also perturbed the transport of spermatids and other organelles (such as phagosomes) across the epithelium. These changes thus led to disruption of spermatogenesis. Interestingly, the knockdown of dynein 1 or its inactivation by ciliobrevin D also perturbed gross disruption of F-actin across the Sertoli cells in vitro and the seminiferous epithelium in vivo, illustrating there are cross talks between the two cytoskeletons in the testis. In summary, these findings confirm the role of cytoplasmic dynein 1 to support the transport of spermatids and organelles across the seminiferous epithelium during the epithelial cycle of spermatogenesis.

scholarly journals Histochemical identification of the vascular endothelial isoenzyme of alkaline phosphatase.

1989 ◽  
Vol 37 (12) ◽  
pp. 1893-1898 ◽  
Author(s):  
H F Zoellner ◽  
N Hunter
Keyword(s):  
Alkaline Phosphatase ◽  
Physiological Role ◽  
Freeze Substitution ◽  
Differential Sensitivity ◽  
Rat Tissues ◽  
Microvascular Endothelium ◽  
Membrane Bound ◽  
Specific Inhibitors

Alkaline phosphatase (AP) is a widely studied membrane bound ecto-enzyme with an extensive distribution in nature. Three major human isoenzymes have been defined and can be distinguished on the basis of their differential sensitivity to specific inhibitors. Despite the voluminous literature describing AP, the physiological role of this enzyme is unclear. Microvascular endothelium is strongly AP positive and may provide a convenient model for study of the role of AP in vitro. This report describes the use of freeze-substitution and high-resolution plastic embedding techniques to identify the isoenzyme of endothelial AP by quantitative analysis of the relative inhibition by specific inhibitors of AP, using human gingival tissues and a number of rat tissues. Endothelial AP is found to be the liver/bone/kidney isoenzyme, indicating kidney as a credible source of enzyme for further experimental work investigating the role of AP.

Sign in / Sign up

Export Citation Format

Share Document