R. C. Youngblood

Application of quantum dot nanoparticles for potential non-invasive bio-imaging of mammalian spermatozoa.

BackgroundVarious obstacles are encountered by mammalian spermatozoa during their journey through the female genital tract, and only few or none will reach the site of fertilization. Currently, there are limited technical approaches for non-invasive investigation of spermatozoa migration after insemination. As the knowledge surrounding sperm behavior throughout the female genital tract still remains elusive, the recent development of self-illuminating quantum dot nanoparticles may present a potential means for real-time in vitro and in vivo monitoring of spermatozoa.ResultsHere, we show the ability of boar spermatozoa to harmlessly interact and incorporate bioluminescent resonance energy transfer-conjugated quantum dot (BRET-QD) nanoparticles. The confocal microscope revealed in situ fluorescence of BRET-QD in the entire spermatozoon, while the ultra-structural analysis using the transmission electron microscope indicated BRET-QD localization on the sperm plasma membrane and intracellular compartment. In controlled-in vitro assays, bioluminescent imaging demonstrated that spermatozoa incubated with BRET-QD and luciferase substrate (coelenterazine) emit light (photons/sec) above the background, which confirmed the in situ fluorescence imaging. Most importantly, sperm motility, viability, and fertilizing potential were not affected by the BRET-QD incorporation when used at an appropriated ratio.ConclusionsOur results demonstrate that pig spermatozoa can incorporate BRET-QD nanoparticles without affecting their motility and capacity to interact with the oocyte when used at an appropriated balance. We anticipate that our study will enable in-depth exploration of the male components of in vivo migration, fertilization, and embryonic development at the molecular level using this novel approach.

Photonic Monitoring in Real Time of Vascular Endothelial Growth Factor Receptor 2 Gene Expression under Relaxin‐Induced Conditions in a Novel Murine Wound Model

Abstract: Relaxin is known to promote vascular endothelial growth factor (VEGF) expression in reproductive tissue, and successful wound healing depends on good vascularization of wound sites, a process that relaxin may facilitate. Thus, the objective of this study was to evaluate the efficacy of relaxin on the development of vascular tissue at wound sites in a novel VEGF receptor 2‐luc (VEGFR2‐luc) transgenic mouse wound model by monitoring the rate of VEGFR2‐luc‐mediated gene expression using bioluminescence and real‐time imaging. To this end, 12 FVB/N VEGFR2‐luc transgenic male mice were assigned to treatments (six per group): saline alone or relaxin (1 g/6 h/14 days) administered intraperitoneally (i.p.). On day 0, a set of full‐thickness wounds (6‐mm punch) were generated under anesthesia on the dorsal aspect of each mouse. Photonic emissions were recorded (5‐min collection of photons) from wound sites 10 min after the administration of luciferin (150 mg/kg i.p.) on day 0 and on days 1, 2, 4, 7, 9, 11, and 14 postwounding to quantify luciferase activity using an IVIS 100 biophotonic imaging system. Animals were sacrificed (three per group) on day 7 or 14, and wound tissue specimens were recovered for molecular and histologic analyses. Although photonic emission from wound sites increased (P <.001) over time with peak values obtained by day 7, no significant (P >.05) effect of relaxin treatment on VEGFR2‐luc gene expression was noted at wound sites. Whereas measuring relaxins effect on angiogenesis indirectly via the VEGFR2 model was not successful, photonic imaging provides an exciting new tool using alternative models (i.e., VEGF‐luc mouse) to study relaxin‐induced gene expression in normal (i.e., wound healing) or tumorigenic tissues in real time.

The use of a whole animal biophotonic model as a screen for the angiogenic potential of estrogenic compounds.

Background: Vascular endothelial growth factor (VEGF) is essential for normal vascular growth and development during wound repair. VEGF is estrogen responsive and capable of regulating its own receptor, vascular endothelial growth factor receptor-2 (VEGFR-2). Several agricultural pesticides (e.g., methoxychlor) have estrogenic potential that can initiate inappropriate physiological responses in estrogenic-sensitive tissues following exposure in vivo. Thus, the current study was designed to determine whether the VEGFR-2-Luciferase (Luc) reporter transgenic mouse is a useful model for evaluating estrogenic tendencies of methoxychlor by monitoring wound healing via VEGFR-2-mediated gene expression using bioluminescence and real-time imaging technology. Results: VEGFR-2-Luc gene activity peaked by d 7 (P<0.001) in all groups but was not different (P>0.05) between control and estrogen/methoxychlor exposed mice. Conclusions: Changes in VEGFR-2-Luc gene activity associated with the dermal wound healing process were able to be measured via photonic emission. The increase in vasculature recruitment and formation is paralleled by the increase of VEGFR-2-Luc activity with a peak on day 7. However, estrogen/methoxychlor did not significantly alter wound healing mediated VEGFR-2-Luc gene expression patterns compared to controls. This suggests that the VEGFR-2-Luc transgenic mouse wound model tested in this study may not be optimal for use as a screen for the angiogenic potential of estrogenic compounds.