Microtubule and microfilament organization in immature, in vitro matured and in vitro fertilized prepubertal goat oocytes

Zygote ◽  
2005 ◽  
Vol 13 (2) ◽  
pp. 155-165 ◽  
Author(s):  
Esther Velilla ◽  
Elisabet Rodríguez-Gonzalez ◽  
Francesca Vidal ◽  
Maria-Teresa Paramio
Keyword(s):  
Laser Scanning ◽  
Polar Body ◽  
Germinal Vesicle ◽  
Fluorescein Isothiocyanate ◽  
Confocal Laser Scanning Microscope ◽  
Meiotic Spindle ◽  
Confocal Laser ◽  
Cortical Region ◽  
Microtubule Networks

The aim of our study was to analyse the cytoskeletal organization of prepubertal goat oocytes. Microtubule and microfilament organization during in vitro maturation of prepubertal and adult goat oocytes and presumptive zygotes of in vitro matured–in vitro fertilized (IVM-IVF) prepubertal goat oocytes were analysed. Oocytes were matured in M-199 with hormones and serum and inseminated with frozen-thawed spermatozoa. Oocytes and presumptive zygotes were treated with anti-α-tubulin antibody and fluorescein isothiocyanate (FITC)-labelled goat anti-mouse antibody to stain the microtubules. Microfilaments were localized by means of phalloidin 5 μg/ml conjugated with fluorescein isothiocyanate (FITC-phalloidin). DNA was stained with propidium iodide. Stained oocytes were observed under a confocal laser scanning microscope. At the germinal vesicle nuclear stage, microfilaments were distributed at the cortex of the oocytes. After in vitro maturation, 91.7% of metaphase II (MII) oocytes from adult goats displayed microfilaments in the cortex and within the polar body and were characterized by the presence of a microfilament thickening at the cortical region over the meiotic spindle. In prepubertal goat MII oocytes only 5.7% of oocytes displayed microfilaments at the cortex and within the polar body. After insemination, most of the zygotes displayed microfilaments distributed at the cortex. An undefined microtubular network was observed in adult and prepubertal goat oocytes at the germinal vesicle stage. After in vitro maturation, 100% of MII oocytes from adult goats displayed microtubules on the meiotic spindle and within the polar body. This pattern of distribution was observed in 71.6% of prepubertal goat oocytes. Undefined microtubule networks were present in most of the zygotes analysed. In conclusion, cytoskeletal differences were found between prepubertal and adult goat MII oocytes. Furthermore, most of the zygotes from IVM-IVF prepubertal goat oocytes displayed cytoskeletal anomalies.

scholarly journals Proline-rich tyrosine kinase2 is involved in F-actin organization during in vitro maturation of rat oocyte

Reproduction ◽  
2006 ◽  
Vol 132 (6) ◽  
pp. 859-867 ◽  
Author(s):  
Xiao-Qian Meng ◽  
Ke-Gang Zheng ◽  
Yong Yang ◽  
Man-Xi Jiang ◽  
Yan-Ling Zhang ◽  
...  
Keyword(s):  
Oocyte Maturation ◽  
Actin Filaments ◽  
Laser Scanning ◽  
Polar Body ◽  
Laser Scanning Microscopy ◽  
Meiotic Spindle ◽  
Confocal Laser ◽  
Cell Cortex ◽  
First Polar Body

Microfilaments (actin filaments) regulate various dynamic events during meiotic maturation. Relatively, little is known about the regulation of microfilament organization in mammalian oocytes. Proline-rich tyrosine kinase2 (Pyk2), a protein tyrosine kinase related to focal adhesion kinase (FAK) is essential in actin filaments organization. The present study was to examine the expression and localization of Pyk2, and in particular, its function during rat oocyte maturation. For the first time, by using Western blot and confocal laser scanning microscopy, we detected the expression of Pyk2 in rat oocytes and found that Pyk2 and Try402 phospho-Pyk2 were localized uniformly at the cell cortex and surrounded the germinal vesicle (GV) or the condensed chromosomes at the GV stage or after GV breakdown. At the metaphase and the beginning of anaphase, Pyk2 distributed asymmetrically both in the ooplasm and the cortex with a marked staining associated with the chromosomes and the region overlying the meiotic spindle. At telophase, Pyk2 was observed in the cleavage furrows in addition to its cortex and cytoplasm localization. The dynamics of Pyk2 were similar to that of F-actin, and this kinase was found to co-localize with microfilaments in several developmental stages during rat oocyte maturation. Microinjection of Pyk2 antibody demolished the microfilaments assembly and also inhibited the first polar body (PB1) emission. These findings suggest an important role of Pyk2 for rat oocyte maturation by regulating the organization of actin filaments.

scholarly journals Grape Seed Extract as a Potential Remineralizing Agent: A Comparative in vitro Study

2012 ◽  
Vol 13 (4) ◽  
pp. 425-430 ◽  
Author(s):  
Shiny Benjamin ◽  
Roshni LNU ◽  
Sabeena Susan Thomas ◽  
Mohan Thomas Nainan
Keyword(s):  
Laser Scanning ◽  
Grape Seed Extract ◽  
Grape Seed ◽  
Confocal Laser Scanning Microscope ◽  
Seed Extract ◽  
Confocal Laser ◽  
Scanning Microscope ◽  
Caries Lesions ◽  
Calcium Glycerophosphate

ABSTRACT Objective Remineralization is an effective treatment that may stop or reverse early tooth decay. Grape seed extract (GSE) is the potential remineralizing agent under investigation. Materials and methods Sound human tooth sections were obtained from the cervical portion of the root and stored in demineralizing solution at 37°C for 96 hours to induce artificial root caries lesions. The sections were divided into four treatment groups including 6.5% grape seed extract, sodium monofluorophosphate (220 ppm) with 0.05% calcium glycerophosphate, 0.5% calcium glycerophosphate and control (no treatment). An in vitro pH cycling model was used to cycle the demineralized specimens through treatment solutions, acidic buffer and neutral buffer for 8 days at 6 cycles per day. Subsequently, they were evaluated using confocal laser scanning microscope. Data were analyzed using analysis of variance (p < 0.05). Results GSE revealed less demineralization and more remineralization compared with other groups. Conclusion GSE promotes remineralization of artificial root caries lesions. Clinical significance The search for the perfect remineralizing agent continues to this day. GSE could be a welcome addition to the remineralization armamentarium. Abbreviations and acronyms GSE: Grape seed extract; ppm: Parts per million; CaGP: Calcium glycerophosphate; CLSM: Confocal laser scanning microscope; ANOVA: Analysis of variance; PA: Proanthocyanidin; CEJ: Cementoenamel junction; mM: Millimole; CaCl2.2H2O: Calcium chloride dihydrate; KH2PO4: Potassium dehydrate phosphate; K2HPO4: Dipotassium phosphate; dH2O: Deionized water; w/v: Weight by volume; ROD: Relative optical density; nm: Nanometer; SD: Standard deviation. How to cite this article Benjamin S, Roshni, Thomas SS, Nainan MT. Grape Seed Extract as a Potential Remineralizing Agent: A Comparative in vitro Study. J Contemp Dent Pract 2012;13(4):425-430.

Synthesis, singlet oxygen generation, photocytotoxicity and subcellular localization of azobisporphyrins as potentially photodynamic therapeutic agents in vitro cell study

2017 ◽  
Vol 21 (02) ◽  
pp. 122-127 ◽  
Author(s):  
Yunman Zheng ◽  
Sizhe Zhu ◽  
Lijun Jiang ◽  
Fengshou Wu ◽  
Chi Huang ◽  
...  
Keyword(s):  
Singlet Oxygen ◽  
Hela Cells ◽  
Cellular Localization ◽  
Laser Scanning ◽  
Laser Scanning Confocal Microscopy ◽  
Confocal Laser Scanning Microscope ◽  
Confocal Laser ◽  
Oxygen Generation ◽  
Scanning Confocal Microscopy

Three azobisporphyrins (Por1, Por2 and Por3) were synthesized by coupling two molecules of (4-nitrophenyl/pyridyl) porphyrins in the presence of KOH/butanol. The structures of porphyrins were confirmed by UV, IR, NMR and mass spectra and elemental analysis. With tetraphenylporphyrin (H2TPP) as a control, the singlet oxygen (1O[Formula: see text] generation of porphyrins was evaluated through 1,3-diphenylisobenzofuran (DPBF) method. The order of ability to generate 1O2 for three azobisporphyrins was Por 1 [Formula: see text]Por 2 > Por 3[Formula: see text] H2TPP. The photocytotoxicity and sub-cellular localization of azobisporphyrins over Hela cells were studied through MTT analysis and confocal laser scanning microscope, respectively. The results indicated Por 1 and Por 2 displayed the low dark-cytotoxicity, while Por 3 induced a concentration-dependent cytotoxicity to Hela cells with the concentration of porphyrins ranging from 1 to 100 [Formula: see text] M. With the light dose at 4 J/cm2, Por 3 killed more than 60% Hela cells at 2 [Formula: see text] M, indicating a high photocytoxicity. As seen from the laser scanning confocal microscopy images, Por 3 was mainly localized in cell membrane, while Por 1 and Por 2 do not displayed significant fluorescent emission in Hela cells. These results suggest the synthesized cationic azobisporphyrin could be used as a potential therapeutic agent for photodynamic therapy of cancers.

A Facile Synthesis of Acid-Activatable Nanoparticles for Efficient Intracellular Doxorubicin Release

2017 ◽  
Vol 46 ◽  
pp. 20-30 ◽  
Author(s):  
Cao Ming ◽  
Xiao Wan Song ◽  
Yu Jiao Zhang ◽  
Chang Zhi Xu ◽  
Peng Chen ◽  
...  
Keyword(s):  
Drug Delivery ◽  
Hela Cells ◽  
Laser Scanning ◽  
Human Cancer ◽  
Polymeric Nanoparticles ◽  
Confocal Laser Scanning Microscope ◽  
Ph Sensitive ◽  
Confocal Laser ◽  
Cell Nuclei

pH responsive polymeric nanoparticles have emerged as a promising technology platform for targeted and controlled drug delivery in recent years. In this paper, endosomal pH-activatable doxorubicin (DOX) and core-crosslinked polymeric nanoparticles (DCNPs) were prepared and investigated for potent growth inhibition of human cancer cells in vitro. In vitro drug release studies, DOX conjugated nanoparticles with hydrazone bond showed a pH sensitive release phenomenon, that is, the releasing is significantly faster at mildly acidic condition with pH of 5.5 than that at physiological condition. Confocal laser scanning microscope (CLSM) observations revealed that DOX conjugated nanoparticles delivered and released DOX into the cytosols as well as cell nuclei of Hela cells following 6 h incubation. MTT assays demonstrated that these pH-sensitive DOX nanoparticles exhibited high antitumor effect to HeLa cells. The conjugated DOX polymeric nanoparticles may be a promising candidate as a nanoscale and pH-sensitive drug delivery vehicle for cancer therapy.

scholarly journals Increased mitochondrial distribution in early-cleaving embryos indicate successful pre-implantation development

2018 ◽  
Vol 14 (4) ◽  
pp. 512-514
Author(s):  
Nor Shahida Abdul Rahman ◽  
Mimi Sophia Sarbandi ◽  
Wan Hafizah Wan Jusof ◽  
Zolkapli Eshak ◽  
Salina Othman ◽  
...  
Keyword(s):  
Laser Scanning ◽  
Close Association ◽  
Mitochondrial Fission ◽  
Polar Body ◽  
Blastocyst Stage ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Cell Stage ◽  
Second Polar Body

The timing of the first zygotic cleavage is an accurate predictor of embryo quality. Embryos that cleaved early have higher developmental viability compared to their late counterparts. During embryonic development, cleavage is affected by cellular metabolic processes performed by mitochondria and its synergistic interaction with endoplasmic reticulum (ER). However, in depth study on differences of mitochondria and ER ultrastructures in early- cleaving (EC) versus late- cleaving (LC) embryos is limited. This study compares mitochondria and ER ultrastructures of EC versus LC embryos using Confocal Laser Scanning Microscopy (CLSM) and Transmission Electron Microscopy (TEM). Embryos were obtained from female ICR superovulated mice, 28-30 hours post hCG. Two-cell embryos were categorized as early-cleaving (EC), while zygotes with the second polar body and two pronuclei present were categorized as late-cleaving (LC). The LC embryos were cultured in vitro until the 2- cell stage. In EC embryos, mitochondria were mostly found at the perinuclear region and closely associated with dense ER. Meanwhile, mitochondria of LC embryos were distributed uniformly within the cytoplasm. Mitochondrial fluorescence intensity was significantly higher in EC versus LC [(18.7 ± 0.4) versus (14.6 ± 0.4)] x 105 pixel, (p<0.01). Development to the blastocyst stage was also significantly higher in EC compared to LC embryos (96.7% versus 60.9%) (p<0.01). Higher viability of EC embryos is attributed to the close association of their mitochondria to ER. This contributed to better mitochondrial fission, resulting in enhanced energy generating processes and preimplantation development. 

scholarly journals Mitochondrial function in vitrified versus slow-frozen murine embryos

2019 ◽  
Vol 15 (2) ◽  
pp. 150-152
Author(s):  
Razif Dasiman ◽  
Mimi-Sophia Sarbandi ◽  
Nor-Shahida Abdul Rahman ◽  
Salina Othman ◽  
Mastura Malek ◽  
...  
Keyword(s):  
Mitochondrial Function ◽  
Laser Scanning ◽  
Developmental Stages ◽  
Confocal Laser Scanning Microscope ◽  
Slow Freezing ◽  
Confocal Laser ◽  
Mitochondrial Functions ◽  
Murine Embryos ◽  
Cryopreserved Embryos

The effects of vitrification and slow-freezing on mitochondrial functions of in vitro produced murine embryos at various developmental stages were investigated using the Confocal Laser Scanning Microscope (CLSM). Oocytes were obtained from superovulated females, fertilized with sperm and cultured. Resulting 2-, 4- and 8-cell embryos were collected and cryopreserved by vitrification and slow-freezing. Mitochondria were stained with MitoTracker Red (CMXRos). Images were viewed by CLSM and analyzed using QWin SoftwareV.3. Fluorescent intensities were used to indicate viability. Results showed that mitochondrial fluorescence intensities of cryopreserved embryos were significantly lower as compared to non-cryopreserved embryos (p<0.01). Vitrification was found to be superior to slow-freezing at all developmental stages, based on mitochondrial function.

scholarly journals 286SPERM-OOCYTE INTERACTION DURING IN VITRO FERTILIZATION IN THE HORSE

2004 ◽  
Vol 16 (2) ◽  
pp. 263
Author(s):  
J.L. Tremoleda ◽  
T.A.E. Stout ◽  
B.M. Gadella ◽  
B. Colenbrander
Keyword(s):  
In Vitro Fertilization ◽  
Laser Scanning ◽  
Transvaginal Ultrasound ◽  
Polar Body ◽  
Molecular Probes ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Vitro Fertilization

In vitro fertilization (IVF) has proven to be a surprisingly unsuccessful way of producing horse embryos. The aim of this study was to investigate the interaction between sperm and the cumulus oocyte complex (COC) during IVF. In experiment 1, three IVF conditions were tested: (A) COCs recovered from slaughtered mares were categorized with respect to cumulus morphology (C: compact, n=86, or E: expanded, n=55) and matured in TCM199 containing 0.01IU/mL porcine FSH and equine LH (IVM); after IVM, the oocytes were denuded and those with a visible polar body were incubated with sperm (IVF) in the presence or absence of 150ng/mL progesterone (P4) to induce the acrosome reaction (AR); (B) IVM oocytes from C-COCS were denuded (n=52) or not (n=67) before IVF in the presence of P4;; (C) in vivo-matured oocytes (n=15) recovered by transvaginal ultrasound-guided aspiration from preovulatory follicles 32h after the donor mare was treated with hCG, were fertilized in vitro in the presence of P4. In all cases, IVF was performed with frozen-thawed, Percoll-selected sperm from a single stallion, at a final concentration of 1×106spermatozoa/ml in fertil-TALP for 20h (Parrish et al., 1988 Biol. Reprod. 38, 1171–1180). In experiment 2, the possibility that semen cryopreservation or stallion critically influenced IVF was examined by incubating denuded IVM oocytes with fresh or frozen/thawed sperm from the same (fresh;; n=17 for both C- and E-COCs and frozen-thawed; n=12 and 21 for C and E-COCs, respectively) or one other stallion (Fresh;; n=12 and 19 and frozen-thawed; n=12 and 19 for C and E-COCs, respectively), in the presence of P4 for 20h. In both experiments, the resulting sperm-oocyte complexes were fixed, permeabilized and labelled with fluorescein-conjugated peanut agglutinin (EY Laboratores, San Mateo, CA, USA) and ethidium homodimer (Molecular Probes, Eugene, OR, USA) to stain the acrosomal membrane and DNA, respectively, so that membrane status and position of the sperm within the oocyte investments could be detected by confocal laser scanning microscopy. The total number of sperm bound per oocyte was compared between treatments using one-way ANOVA with pair-wise multiple comparison (Bonferroni t-test). Despite binding to the zona pellucida (ZP), neither fresh nor frozen/thawed sperm from either stallion acrosome-reacted or penetrated any oocytes, irrespective of cumulus morphology at the onset of IVM, denudation prior to IVF or the presence of P4. However, more sperm bound to the ZP of cumulus-denuded IVM oocytes (65±32 and 62±28 [mean±sd] for C and E-COCs, respectively), than cumulus-intact IVM (5±4) or in vivo-matured oocytes (23±17: P&lt;0.001). None of the other factors investigated affected bound sperm numbers. In all cases, ZP-bound sperm failed to AR in the classical fashion, and all oocytes remained arrested at the MII stage. In summary, fertilization failed because sperm did not acrosome-react after binding to the ZP. It is concluded that failure to adequately activate stallion sperm is an important obstacle to successful IVF in horses.

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