title: Self-illuminating quantum dots for non-invasive bioluminescence imaging of mammalian gametes.
abstract: 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 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.
  
  
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Animal and Dairy Sciences, Facility for Organismal and Cellular Imaging (FOCI), Mississippi State University, Mississippi State, MS, 39762, USA. jn181@ads.msstate.edu.
authors.forename: Jean M
authors.initials: JM
authors.lastname: Feugang
authors.affiliation: Department of Animal and Dairy Sciences, Facility for Organismal and Cellular Imaging (FOCI), Mississippi State University, Mississippi State, MS, 39762, USA. rcy7@msstate.edu.
authors.forename: Ramey C
authors.initials: RC
authors.lastname: Youngblood
authors.affiliation: Department of Animal and Dairy Sciences, Facility for Organismal and Cellular Imaging (FOCI), Mississippi State University, Mississippi State, MS, 39762, USA. jmicg211@gmail.com.|Department of Pathobiology and Population Medicine, Mississippi State University, Mississippi State, MS, 39762, USA. jmicg211@gmail.com.
authors.forename: Jonathan M
authors.initials: JM
authors.lastname: Greene
authors.affiliation: Department of Animal and Dairy Sciences, Facility for Organismal and Cellular Imaging (FOCI), Mississippi State University, Mississippi State, MS, 39762, USA. swillard@cals.msstate.edu.|Department of Biochemistry and Molecular Biology and Entomology and Plant Pathology, Mississippi State University, Mississippi State, MS, 39762, USA. swillard@cals.msstate.edu.
authors.forename: Scott T
authors.initials: ST
authors.lastname: Willard
authors.affiliation: Department of Animal and Dairy Sciences, Facility for Organismal and Cellular Imaging (FOCI), Mississippi State University, Mississippi State, MS, 39762, USA. ryan@provost.msstate.edu.|Department of Pathobiology and Population Medicine, Mississippi State University, Mississippi State, MS, 39762, USA. ryan@provost.msstate.edu.
authors.forename: Peter L
authors.initials: PL
authors.lastname: Ryan
mesh_terms: D000818:Animals; D002470:Cell Survival; D005260:Female; D005836:Genitalia, Female / Q000502:physiology; D049410:Luciferases, Renilla / Q000737:chemistry; D049408:Luminescent Agents / Q000737:chemistry / Q000493:pharmacokinetics*; D008163:Luminescent Measurements / Q000295:instrumentation / Q000379:methods*; D008297:Male; D018613:Microscopy, Confocal / Q000379:methods; D046529:Microscopy, Electron, Transmission; D053758:Nanoparticles / Q000737:chemistry; D009865:Oocytes / Q000737:chemistry / Q000502:physiology*; D006080:Ovarian Follicle / Q000502:physiology; D045663:Quantum Dots; D013094:Spermatozoa / Q000737:chemistry / Q000502:physiology*; D013552:Swine
chemical_list: D049408:Luminescent Agents; D049410:Luciferases, Renilla