Application of quantum dot nanoparticles for potential non-invasive bio-imaging of mammalian spermatozoa
© Feugang et al.; licensee BioMed Central Ltd. 2012
Received: 30 September 2012
Accepted: 11 December 2012
Published: 14 December 2012
Various 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.
Here, 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.
Our 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.
KeywordsSpermatozoa Fertilization Quantum dot Nanoparticles Biophotonic imaging Bioluminescence imaging
Mammalian spermatozoa are tiny and highly specialized cells shaped to enable migration through the female genital tract, interact with oocytes, and deliver the paternal materials to the oocyte. However, various obstacles associated with spermatozoa themselves or encountered within the female genital tract may lead to few or no spermatozoa reaching the site of fertilization[1–3]. This situation inevitably affects the pregnancy outcome and there is a crucial need to better understand the sperm behavior within the female genital tract (i.e., migration and interactions with its surrounding environment), as well as the molecular and cellular events that precede fertilization in vivo.
At present, the conventional experimental approaches of studying mammalian spermatozoa are limited by researchers’ inability to accurately and non-invasively investigate sperm quality and viability before and after insemination[4–7], as the normal sperm progression within the female genital tract remains unclear. The development of new techniques that enable non-invasive monitoring of sperm movement after artificial insemination has the potential to enhance breeding efficiencies, which could be achieved by either selecting sires with spermatozoa more apt to overcome utero-oviductal hindrances and encounter oocytes in vivo or dams that are more likely to facilitate the migration and interaction of both gametes. A recent study towards this goal has successfully imaged living ram spermatozoa in different settings (in vitro, ex vivo, and in vivo) using organic fluorochromes, which have limitations in terms of brightness and photo-stability for deep-tissue imaging[8, 9].
As an alternative to traditional organic fluorescent dyes, such as green fluorescence protein, the recent progress in the nanotechnology field has led to the production of biocompatible quantum dot (QD) nanoparticles that are highly photo-stable and brighter. These nanoparticles can be produced in various sizes to emit a vast spectra of wavelengths upon a single excitation and, therefore, permit their utilization in various areas of biomedicine for targeted and non-targeted in vitro and in vivo imaging[10, 12–14]. Most interestingly, the ability of QD to fluoresce in the near infra-red spectrum and to be linked to a variety of substances (i.e., peptides, nucleic acids, and luciferase) creates more opportunities for these nanoparticles[11, 15, 16]. At present, the nanotechnology has not been applied in the field of reproductive biology while it could be useful for molecular imaging. We believe this technology can provide invaluable insight into biological and cellular processes associated with gamete behavior and interactions, and early embryo development.
In this study, we explored the potential use of QD nanoparticles as a flexible tool to apply for non-invasive investigation of mammalian spermatozoa. Quantum dots emitting at 655 nm wavelength and conjugated with Renilla luciferase and nona-arginine R9 internalization peptide (BRET-QD;) were used to label boar spermatozoa, followed by the assessment of their impact on sperm motility, viability, and fertilizing potential.
Results and discussion
Here, we investigated the ability of mammalian spermatozoa to harmlessly incorporate CdSe/ZnS QD nanoparticles conjugated to the nona-Arginine R9 peptide that facilitates its cellular internalization. For bio-imaging purpose, QD were linked to the Renilla luciferase enzyme which in the presence of its substrate, coelenterazine, creates a self-illuminating QD-Bioluminescent Resonance Energy Transfer complex (BRET-QD) emitting both light and fluorescence that are captured with appropriate equipment.
Evaluation of BRET-QD internalization in spermatozoa
Bioluminescence imaging of spermatozoa exposed to BRET-QD
Altogether, our data indicate that large amounts of BRET-QD can interact with living mammalian spermatozoa, which is of great interest for in vivo imaging. Nonetheless, the ability of these nano-sized particles to enter cells may cause unexpected toxicities which have already been reported in somatic cells[28, 29].
Assessment of BRET-QD internalization on sperm viability and fertilizing potential
As a first step to assess the potential toxicity of BRET-QD, we evaluated the motility, viability and fertilizing potential of spermatozoa after incubation (30 min) with BRET-QD.
Effect of BRET-QD and sperm ratio on sperm motility
87 ± 6a
74 ± 5
94 ± 11
202 ± 23a
56 ± 9
0.1 x 108
36 ± 6b
23 ± 13
40 ± 5
86 ± 15b
21 ± 5
0.5 x 108
63 ± 17ab
46 ± 15
65 ± 19
143 ± 36ab
39 ± 12
1 x 108
81 ± 8a
68 ± 11
90 ± 9
194 ± 17a
51 ± 7
2 x 108
89 ± 5a
63 ± 22
94 ± 17
198 ± 36a
52 ± 8
P values (ANOVA 2)
Effect of BRET-QD on sperm viability
Proportions of spermatozoa with intact:
exposed to BRET-QD at:
Plasma membrane (%)
Mitochondrial membrane (%)
77.0 ± 3.3
95.8 ± 3.5
78.8 ± 2.0
97.4 ± 1.7
77.3 ± 2.3
97.0 ± 2.4
P values (ANOVA-2)
P = 0.867
P = 0.906
Fertilizing potential of BRET-QD labeled spermatozoa
Proportion (%) of total oocytes analyzed as:
Total number of oocytes
63 ± 7
37 ± 7
59 ± 9
41 ± 4
As the main function of the spermatozoa is to deliver the paternal materials to the oocyte, we further investigated the capability of BRET-QD labeled-spermatozoa to fertilize the oocyte. Based on the motility data above, we used spermatozoa (108) labeled with 1 nM BRET-QD to fertilize in vitro-matured pig oocytes at a final concentration of 6x105 sperm/ml. Table3 shows comparable proportions of fertilized oocytes with unlabeled (control) and labeled (exposed) spermatozoa (63% ± 7% and 59% ± 9%, respectively; P > 0.05). These results indicated that the sperm labeling with sufficient amount of BRET-QD does not affect their fertilizing potential. Although the developmental performance of fertilized oocytes was not evaluated in our study, a recent report has demonstrated that exposure of oocytes to higher concentrations of CdSe core (125 nM) or CdSe/ZnS core-shell (500 nM) do not affect their developmental competence (fertilization, developmental and implantation rates and reduction of apoptosis and cell proliferation in blastocysts).
This study reports the possibility to label mammalian spermatozoa with bioluminescence resonance energy transfer CdSe/ZS quantum dot linked to the arginine-rich cell penetrating peptide R9. The results suggest that the self-illuminating BRET-QD can be employed for molecular imaging in mammalian spermatozoa without causing functional interference. Our results lay the ground work for implementing novel imaging techniques that can be utilized both for exploring important molecular characteristics of spermatozoa and for in vivo tracking of labeled-spermatozoa through a fluorescence endo-microscopy approach. The application of such imaging technology will allow a better understanding of sperm migration within the female genital tract.
Materials and reagents
A stock solution of CdSe/ZnS core-shell structure quantum dots (500 nM in Tris buffer) cross-linked to Renilla luciferase (BRET) and nona-arginine R9 peptide was purchased from Zymera Inc. (San Jose, CA, USA). The BRET-QD complex is a self-illuminating nanoparticle that emits light under incubation with coelenterazine (luciferase substrate; Zymera Inc.), and exhibits intense fluorescence with red-shifted emission (655 nm) following excitation. Boar semen was obtained from Prestage Farms (West Point, MS, USA) and oocytes from post-mortem gilt ovaries (South Quality Meats, Pontotoc, MS, USA).
Sperm preparation and loading with BRET-QD
Freshly collected motile boar spermatozoa were selected as previously. Spermatozoa (2 x 108 sperm/ml) were incubated with 0, 1, or 5 nM BRET-QD at 37°C for 30 min. After three washes by centrifugation (1,000 g – 3 min) with PBS-PVP (1 mg/ml), supernatants containing excess QD were removed and 50 μl of each were kept for bioluminescence imaging. In parallel, sperm pellets were suspended with 50 μl PBS-PVP for experiments.
A total of 4 μg of coelenterazine was added to each cell suspensions and supernatants. All samples were imaged within around 5 min, but less than 10 min (photons/sec) using the IVIS 100 bioluminescence imager system (Caliper Life Sciences, Hopkinton, MA) with a 1 min acquisition time and without any filter.
Detection of BRET-QD fluorescence emission within spermatozoa
Aliquots of spermatozoa incubated with 0, 1 or 5 nM BRET-QD were mounted onto microscope slides to evaluate their fluorescence emission. Samples were analyzed under a Laser Scanning Microscope system (LSM510, Carl Zeiss Micro Imaging GmbH, Jena, Germany) with a 488 nm excitation and 660/20 nm emission. The background fluorescence of samples without BRET-QD served as controls.
BRET-QD localization in spermatozoa
Spermatozoa exposed to 0, 1, or 5 nM BRET-QD were suspended in phosphate-buffered 2.5% glutaraldehyde fixative solution. The standard protocol for sample preparation for transmission electron microscopy (TEM-JEOL) was performed without osmium tetroxide fixation and uranyl acetate staining. Here, we excluded both steps in order to increase the background contrast and BRET-QD signal, and prepared samples of pure BRET-QD were placed on formvar-coated grids for TEM analysis. In parallel, aliquots of BRET-QD were placed on coated-slides to evaluate BRET-QD using Atomic Force Microscope (AFM).
Sperm motility and viability analyses
Immediately after incubation of spermatozoa and removal of the excess of BRET-QD, aliquots of spermatozoa were submitted to the motion analysis using a Computer Assisted Sperm Analyzer (CASA; IVOS v12; Hamilton Thorne Biosciences, Beverly, MA, USA). Motility characteristics of spermatozoa were determined using 20 μm4-chamber glass counting slides (Leja Products, Nieuw-Vennep, The Netherlands).
Additional aliquots of labeled or non-labeled spermatozoa were used for viability analyses after staining of cells for either plasma (Propidium Iodide; 10 μg/ml; Sigma-Aldrich Co., Saint Louis, MO, USA) or mitochondrial (JC-1; Cayman Chemical Co., Ann Arbor, MI, USA) membrane integrities. The proportions of viable spermatozoa were evaluated with a flow cytometer (Becton Dickinson FACSCalibur) set for 10,000 total events per analysis.
Fertilizing potential of spermatozoa
Ovaries were collected from post-mortem gilts and oocytes were selected and matured in vitro according to Feugang et al.. Matured oocytes were fertilized at a final concentration of 6 × 105 spermatozoa/ml with spermatozoa (108) pre-exposed to 0 or 1 nM BRET-QD diluted in PBS-PVP (1 mg/ml). After 18 h co-incubation, the proportions of fertilized and non-fertilized oocytes were evaluated as previously reported.
Each experiment was repeated at least three times. Fertilization data were analyzed using the z-ratio, to evaluate the significance of the difference between proportions in the control and exposed (1nM BRET-QD) groups. Bioluminescence, motility, viability and velocity data were analyzed using the two-way ANOVA that considered both replicates (N) and groups. Pairwise comparisons were performed using the Fisher’s LSD test. Data are expressed as mean ± s.e.m., unless otherwise indicated. The threshold of significance was set at P ≤ 0.05 and tendency at P < 0.1.
We thank C. I-Wei and A. Lawrence (Institute for Imaging and Analytical Technologies, Mississippi State University) for their assistance with microscopy techniques (Confocal and AFM), J. Stokes (Basic Sciences department, Mississippi State University) for his help on the cell viability analysis, Mr. D. Sobek (Zymera, Inc.) for his assistance on quantum dot analysis, and Dr. M. Crenshaw (Animal and Dairy Sciences department, Mississippi State University) for his assistance in data interpretation. The authors are grateful to Mr. T. Emerson (Prestage Farms, Mississippi) for providing semen. This work was sponsored by the United States Department of Agriculture, Agricultural Research Station (Biophotonics Research Initiative, grant # 58-6402-3-0120).
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