Quantitative bioluminescence imaging of transgene expression in intact porcine antral follicles in vitro

Jung, Song-Yi; Willard, Scott T.

doi:10.1186/1477-7827-12-11

Cited by 4 publications

(4 citation statements)

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“…Although the viability of cells was not verified, data suggest that self-illuminating luminescent nanoparticles can be used for molecular imaging of cultured follicles. Our finding adds to a recent study reporting a successful intra-follicular imaging through a transgene carrying the luciferase reporter gene [ 49 ]. The current work paves the way for further optimization of this novel non-invasive imaging approach that will lead to promising in situ real-time description of the oocyte maturation and related intra-follicular key molecules.…”

Section: Resultssupporting

confidence: 67%

Self-illuminating quantum dots for non-invasive bioluminescence imaging of mammalian gametes

et al. 2015

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BackgroundThe fertility performance of animals is still a mystery and the full comprehension of mammalian gametes maturation and early embryonic development remains to be elucidated. The recent development in nanotechnology offers a new opportunity for real-time study of reproductive cells in their physiological environments. As a first step toward that goal, we evaluated the effectiveness of a fluorescent and luminescent nanoparticle for in vitro and ex vivo imaging of porcine gametes.MethodsFreshly harvested boar sperm were labeled with red-shifted (655 nm) quantum dot nanoparticles conjugated (QD+) or not (QD−) with plasminogen antibody and evaluated. Subsets of labeled spermatozoa were loaded into straws and placed within the lumen of gilt reproductive tracts for ex vivo intra-uterine imaging. Porcine cumulus-oocyte complexes (COCs) were matured in the presence of QD− or QD+. Ovarian follicles were microinjected with QD− or QD+ and placed in culture for up to 4 days. After labeling, all samples were supplemented with coelenterazine, the luciferase substrate, and immediately submitted to bioluminescence analysis, followed by fluorescence and hyperspectral imaging. Data were analyzed with ANOVA and P < 0.05 indicated significant differences.ResultsAll labeled-samples revealed bioluminescence emission that was confirmed by fluorescence and hyperspectral imaging of the QD localization within the cells and tissues. Over 76% of spermatozoa and both immature and mature COCs were successfully labeled with QD− or QD+. The QD− fluorescence appeared homogenously distributed in the oocytes, while found in the entire sperm length with a higher accumulation within the mid-piece. Labeled-follicles exhibited a progressive migration of QD nanoparticles within the follicle wall during culture. In contrast, QD+ fluorescence signals appeared condensed and stronger in the follicle cells, sperm head, and sub-plasma membrane area of mature oocytes. Weaker QD+ signals were detected in the cumulus cells. Fluorescence and hyperspectral microscope imaging showed comparable intracellular QD localization. Ex-vivo intra-uterine bioluminescence imaging of labeled spermatozoa revealed stronger signals captured over the oviducts, with uterine body allowing the lowest signal detection.ConclusionFindings indicate that conjugated and non-conjugated fluorescent nanoparticles can be used for effective labeling of mammalian gametes for in vitro monitoring and potential in vivo targeted-imaging.Electronic supplementary materialThe online version of this article (doi:10.1186/s12951-015-0097-1) contains supplementary material, which is available to authorized users.

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Section: Resultssupporting

confidence: 67%

Self-illuminating quantum dots for non-invasive bioluminescence imaging of mammalian gametes

et al. 2015

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“…Although the viability of cells was not verified, data suggest that self-illuminating luminescent nanoparticles can be used for molecular imaging of cultured follicles. Our finding adds to a recent study reporting a successful intra-follicular imaging through a transgene carrying the luciferase reporter gene [49]. The current work paves the way for further optimization of this novel non-invasive imaging approach that will lead to promising in situ realtime description of the oocyte maturation and related intra-follicular key molecules.…”

Section: Ovarian Follicle Labeling and Bioluminescence Imagingsupporting

confidence: 58%

Self-illuminating quantum dots for non-invasive bioluminescence imaging of mammalian gametes

Feugang¹,

Youngblood²,

Greene³

et al. 2016

Nano Online

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Background: The fertility performance of animals is still a mystery and the full comprehension of mammalian gametes maturation and early embryonic development remains to be elucidated. The recent development in nanotechnology offers a new opportunity for real-time study of reproductive cells in their physiological environments. As a first step toward that goal, we evaluated the effectiveness of a fluorescent and luminescent nanoparticle for in vitro and ex vivo imaging of porcine gametes. Methods:Freshly harvested boar sperm were labeled with red-shifted (655 nm) quantum dot nanoparticles conjugated (QD+) or not (QD−) with plasminogen antibody and evaluated. Subsets of labeled spermatozoa were loaded into straws and placed within the lumen of gilt reproductive tracts for ex vivo intra-uterine imaging. Porcine cumulusoocyte complexes (COCs) were matured in the presence of QD− or QD+. Ovarian follicles were microinjected with QD− or QD+ and placed in culture for up to 4 days. After labeling, all samples were supplemented with coelenterazine, the luciferase substrate, and immediately submitted to bioluminescence analysis, followed by fluorescence and hyperspectral imaging. Data were analyzed with ANOVA and P < 0.05 indicated significant differences. Results:All labeled-samples revealed bioluminescence emission that was confirmed by fluorescence and hyperspectral imaging of the QD localization within the cells and tissues. Over 76% of spermatozoa and both immature and mature COCs were successfully labeled with QD− or QD+. The QD− fluorescence appeared homogenously distributed in the oocytes, while found in the entire sperm length with a higher accumulation within the mid-piece. Labeled-follicles exhibited a progressive migration of QD nanoparticles within the follicle wall during culture. In contrast, QD+ fluorescence signals appeared condensed and stronger in the follicle cells, sperm head, and sub-plasma membrane area of mature oocytes. Weaker QD+ signals were detected in the cumulus cells. Fluorescence and hyperspectral microscope imaging showed comparable intracellular QD localization. Ex-vivo intra-uterine bioluminescence imaging of labeled spermatozoa revealed stronger signals captured over the oviducts, with uterine body allowing the lowest signal detection. Conclusion: Findings indicate that conjugated and non-conjugated fluorescent nanoparticles can be used for effective labeling of mammalian gametes for in vitro monitoring and potential in vivo targeted-imaging.

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“…Second, in our study, the group with dual p70S6K and eIF4E siRNA, and the rapamycin instillation groups showed lower tumor volume and lower invasiveness than groups with p70S6K or eIF4E siRNA, although no difference was observed between groups in photon densities. Our hypothesis suggests that although the BLI photon density is increased according to tumor volume increase, it has a tendency to be exaggerated ( 27 ); this may be correlated with the factors that influence the intensity of bioluminescent emissions including the time courses of luciferase-luciferin reactions, and effective plasmid DNA and D-luciferin doses and combinations. Although BLI needs to be improved in terms of cost-effectiveness and non-invasiveness, it has many advantages.…”

Section: Discussionmentioning

confidence: 93%

P70S6K and Elf4E Dual Inhibition Is Essential to Control Bladder Tumor Growth and Progression in Orthotopic Mouse Non-muscle Invasive Bladder Tumor Model

et al. 2015

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We investigated how the dual inhibition of the molecular mechanism of the mammalian target of the rapamycin (mTOR) downstreams, P70S6 kinase (P70S6K) and eukaryotic initiation factor 4E (eIF4E), can lead to a suppression of the proliferation and progression of urothelial carcinoma (UC) in an orthotopic mouse non-muscle invasive bladder tumor (NMIBT) model. A KU-7-luc cell intravesically instilled orthotopic mouse NMIBC model was monitored using bioluminescence imaging (BLI) in vivo by interfering with different molecular components using rapamycin and siRNA technology. We then analyzed the effects on molecular activation status, cell growth, proliferation, and progression. A high concentration of rapamycin (10 µM) blocked both P70S6K and elF4E phosphorylation and inhibited cell proliferation in the KU-7-luc cells. It also reduced cell viability and proliferation more than the transfection of siRNA against p70S6K or elF4E. The groups with dual p70S6K and elF4E siRNA, and rapamycin reduced tumor volume and lamina propria invasion more than the groups with p70S6K or elF4E siRNA instillation, although all groups reduced photon density compared to the control. These findings suggest that both the mTOR pathway downstream of eIF4E and p70S6K can be successfully inhibited by high dose rapamycin only, and p70S6K and Elf4E dual inhibition is essential to control bladder tumor growth and progression.Graphical Abstract

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Quantitative bioluminescence imaging of transgene expression in intact porcine antral follicles in vitro

Cited by 4 publications

References 35 publications

Self-illuminating quantum dots for non-invasive bioluminescence imaging of mammalian gametes

Self-illuminating quantum dots for non-invasive bioluminescence imaging of mammalian gametes

Self-illuminating quantum dots for non-invasive bioluminescence imaging of mammalian gametes

P70S6K and Elf4E Dual Inhibition Is Essential to Control Bladder Tumor Growth and Progression in Orthotopic Mouse Non-muscle Invasive Bladder Tumor Model

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