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Internalization of silver nanoparticles into mouse spermatozoa results in poor fertilization and compromised embryo development.

Yoisungnern T, Choi YJ, Han JW, Kang MH, Das J, Gurunathan S, Kwon DN, Cho SG, Park C, Chang WK, Chang BS, Parnpai R, Kim JH - Sci Rep (2015)

Bottom Line: Blastocysts obtained from AgNPs-treated spermatozoa showed lower expression of trophectoderm-associated and pluripotent marker genes.Overall, we propose that AgNPs internalization into spermatozoa may alter sperm physiology, leading to poor fertilization and embryonic development.Such AgNPs-induced reprotoxicity may be a valuable tool as models for testing the safety and applicability of medical devices using AgNPs.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Animal Biotechnology, College of Animal Bioscience and Biotechnology/Animal Resources Research Center, Konkuk University, Seoul 143-701, South Korea [2] Embryo Technology and Stem Cell Research Center, School of Biotechnology, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand.

ABSTRACT
Silver nanoparticles (AgNPs) have many features that make them attractive as medical devices, especially in therapeutic agents and drug delivery systems. Here we have introduced AgNPs into mouse spermatozoa and then determined the cytotoxic effects of AgNPs on sperm function and subsequent embryo development. Scanning electron microscopy and transmission electron microscopy analyses showed that AgNPs could be internalized into sperm cells. Furthermore, exposure to AgNPs inhibited sperm viability and the acrosome reaction in a dose-dependent manner, whereas sperm mitochondrial copy numbers, morphological abnormalities, and mortality due to reactive oxygen species were significantly increased. Likewise, sperm abnormalities due to AgNPs internalization significantly decreased the rate of oocyte fertilization and blastocyst formation. Blastocysts obtained from AgNPs-treated spermatozoa showed lower expression of trophectoderm-associated and pluripotent marker genes. Overall, we propose that AgNPs internalization into spermatozoa may alter sperm physiology, leading to poor fertilization and embryonic development. Such AgNPs-induced reprotoxicity may be a valuable tool as models for testing the safety and applicability of medical devices using AgNPs.

No MeSH data available.


Related in: MedlinePlus

Sperm viability and acrosome reaction analysis.Sperm cells were treated with 0, 0.1, 1, 10, or 50 μg/mL AgNPs. For the ROS inhibitor group, sperm were pretreated with 5 mM NAC for 30 min, and then, 0–50 μg/mL of AgNPs was added. Dead and live sperm cells were counted using the Dead/Live kit. (a) Fluorescence microscopy image. Live sperms cells were stained with SYBR 14 dye (green fluorescence), whereas dead sperm cells were stained with propidium iodide (PI; red fluorescence). Yellow color in the merged image indicates dying sperm cells. (b & c) Dead and live sperm cells were calculated by flow cytometry: FL1 and FL2 represent green (live sperm) and red (dead sperm) color. (d) A representative immunofluorescence staining pattern obtained using mouse anti-CD46 (green) antibody. Nuclei are counterstained with DAPI (blue). (e & f) Flow cytometry analysis of CD46-positive sperm populations. Data for (f) was obtained from the flow cytometry experiment in (e). *p < 0.05, **p < 0.01, and ***p < 0.001 versus the control group (Dunnett’s t-tests).
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f3: Sperm viability and acrosome reaction analysis.Sperm cells were treated with 0, 0.1, 1, 10, or 50 μg/mL AgNPs. For the ROS inhibitor group, sperm were pretreated with 5 mM NAC for 30 min, and then, 0–50 μg/mL of AgNPs was added. Dead and live sperm cells were counted using the Dead/Live kit. (a) Fluorescence microscopy image. Live sperms cells were stained with SYBR 14 dye (green fluorescence), whereas dead sperm cells were stained with propidium iodide (PI; red fluorescence). Yellow color in the merged image indicates dying sperm cells. (b & c) Dead and live sperm cells were calculated by flow cytometry: FL1 and FL2 represent green (live sperm) and red (dead sperm) color. (d) A representative immunofluorescence staining pattern obtained using mouse anti-CD46 (green) antibody. Nuclei are counterstained with DAPI (blue). (e & f) Flow cytometry analysis of CD46-positive sperm populations. Data for (f) was obtained from the flow cytometry experiment in (e). *p < 0.05, **p < 0.01, and ***p < 0.001 versus the control group (Dunnett’s t-tests).

Mentions: As shown in Fig. 2e,f, sperm morphology was examined after incubation with different concentrations of AgNPs. In this study, there were 5 characteristics of spermatozoa: normal, detached head, coiled tails, roll tails, and bent tail (Fig. 2e). We found significant abnormal morphological changes in spermatozoa, such as coiled tail, roll tail, and bent tails at high concentrations of AgNPs (10 μg/mL and 50 μg/mL) (Fig. 2f). The major populations of live spermatozoa that stained with SYBR-14 (green color), dead spermatozoa that stained with propidium iodide (PI; red color), and merged spermatozoa that stained with both green and red (yellow color) are shown in Fig. 3a. Exposure of spermatozoa to AgNPs had a negative effect on sperm viability as measured by flow cytometry analysis. As shown in Fig. 3b, incubation of spermatozoa with AgNPs slightly increased the population of dead spermatozoa (correlation coefficient, r = 0.988). To confirm these results, sperm viability was checked by counting the number of dead and live spermatozoa, which were stained red and green, respectively. These results showed that only at the highest dose, AgNPs exposure significantly (p < 0.05) increased the percentage of dead spermatozoa, whereas the number of live spermatozoa was significantly decreased (Fig. 3c). Since silver ion (Ag+) exposure formed precipitation in the Modified Whitten’s medium, we could not examined whether Ag+ exposure significantly reduced sperm viability. Of note, pre-treatment with NAC for 30 min decreased the AgNPs-induced alterations in sperm viability (Fig. 3c). However, the viability of the spermatozoa cultured in non-capacitation (NCP) medium containing different AgNPs dosages (0.1, 1, 10, and 50 μg/mL AgNPs for 3 h) was more significantly decreased than those cultured in capacitation (CP) medium (Supplementary Fig. 5).


Internalization of silver nanoparticles into mouse spermatozoa results in poor fertilization and compromised embryo development.

Yoisungnern T, Choi YJ, Han JW, Kang MH, Das J, Gurunathan S, Kwon DN, Cho SG, Park C, Chang WK, Chang BS, Parnpai R, Kim JH - Sci Rep (2015)

Sperm viability and acrosome reaction analysis.Sperm cells were treated with 0, 0.1, 1, 10, or 50 μg/mL AgNPs. For the ROS inhibitor group, sperm were pretreated with 5 mM NAC for 30 min, and then, 0–50 μg/mL of AgNPs was added. Dead and live sperm cells were counted using the Dead/Live kit. (a) Fluorescence microscopy image. Live sperms cells were stained with SYBR 14 dye (green fluorescence), whereas dead sperm cells were stained with propidium iodide (PI; red fluorescence). Yellow color in the merged image indicates dying sperm cells. (b & c) Dead and live sperm cells were calculated by flow cytometry: FL1 and FL2 represent green (live sperm) and red (dead sperm) color. (d) A representative immunofluorescence staining pattern obtained using mouse anti-CD46 (green) antibody. Nuclei are counterstained with DAPI (blue). (e & f) Flow cytometry analysis of CD46-positive sperm populations. Data for (f) was obtained from the flow cytometry experiment in (e). *p < 0.05, **p < 0.01, and ***p < 0.001 versus the control group (Dunnett’s t-tests).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4459204&req=5

f3: Sperm viability and acrosome reaction analysis.Sperm cells were treated with 0, 0.1, 1, 10, or 50 μg/mL AgNPs. For the ROS inhibitor group, sperm were pretreated with 5 mM NAC for 30 min, and then, 0–50 μg/mL of AgNPs was added. Dead and live sperm cells were counted using the Dead/Live kit. (a) Fluorescence microscopy image. Live sperms cells were stained with SYBR 14 dye (green fluorescence), whereas dead sperm cells were stained with propidium iodide (PI; red fluorescence). Yellow color in the merged image indicates dying sperm cells. (b & c) Dead and live sperm cells were calculated by flow cytometry: FL1 and FL2 represent green (live sperm) and red (dead sperm) color. (d) A representative immunofluorescence staining pattern obtained using mouse anti-CD46 (green) antibody. Nuclei are counterstained with DAPI (blue). (e & f) Flow cytometry analysis of CD46-positive sperm populations. Data for (f) was obtained from the flow cytometry experiment in (e). *p < 0.05, **p < 0.01, and ***p < 0.001 versus the control group (Dunnett’s t-tests).
Mentions: As shown in Fig. 2e,f, sperm morphology was examined after incubation with different concentrations of AgNPs. In this study, there were 5 characteristics of spermatozoa: normal, detached head, coiled tails, roll tails, and bent tail (Fig. 2e). We found significant abnormal morphological changes in spermatozoa, such as coiled tail, roll tail, and bent tails at high concentrations of AgNPs (10 μg/mL and 50 μg/mL) (Fig. 2f). The major populations of live spermatozoa that stained with SYBR-14 (green color), dead spermatozoa that stained with propidium iodide (PI; red color), and merged spermatozoa that stained with both green and red (yellow color) are shown in Fig. 3a. Exposure of spermatozoa to AgNPs had a negative effect on sperm viability as measured by flow cytometry analysis. As shown in Fig. 3b, incubation of spermatozoa with AgNPs slightly increased the population of dead spermatozoa (correlation coefficient, r = 0.988). To confirm these results, sperm viability was checked by counting the number of dead and live spermatozoa, which were stained red and green, respectively. These results showed that only at the highest dose, AgNPs exposure significantly (p < 0.05) increased the percentage of dead spermatozoa, whereas the number of live spermatozoa was significantly decreased (Fig. 3c). Since silver ion (Ag+) exposure formed precipitation in the Modified Whitten’s medium, we could not examined whether Ag+ exposure significantly reduced sperm viability. Of note, pre-treatment with NAC for 30 min decreased the AgNPs-induced alterations in sperm viability (Fig. 3c). However, the viability of the spermatozoa cultured in non-capacitation (NCP) medium containing different AgNPs dosages (0.1, 1, 10, and 50 μg/mL AgNPs for 3 h) was more significantly decreased than those cultured in capacitation (CP) medium (Supplementary Fig. 5).

Bottom Line: Blastocysts obtained from AgNPs-treated spermatozoa showed lower expression of trophectoderm-associated and pluripotent marker genes.Overall, we propose that AgNPs internalization into spermatozoa may alter sperm physiology, leading to poor fertilization and embryonic development.Such AgNPs-induced reprotoxicity may be a valuable tool as models for testing the safety and applicability of medical devices using AgNPs.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Animal Biotechnology, College of Animal Bioscience and Biotechnology/Animal Resources Research Center, Konkuk University, Seoul 143-701, South Korea [2] Embryo Technology and Stem Cell Research Center, School of Biotechnology, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand.

ABSTRACT
Silver nanoparticles (AgNPs) have many features that make them attractive as medical devices, especially in therapeutic agents and drug delivery systems. Here we have introduced AgNPs into mouse spermatozoa and then determined the cytotoxic effects of AgNPs on sperm function and subsequent embryo development. Scanning electron microscopy and transmission electron microscopy analyses showed that AgNPs could be internalized into sperm cells. Furthermore, exposure to AgNPs inhibited sperm viability and the acrosome reaction in a dose-dependent manner, whereas sperm mitochondrial copy numbers, morphological abnormalities, and mortality due to reactive oxygen species were significantly increased. Likewise, sperm abnormalities due to AgNPs internalization significantly decreased the rate of oocyte fertilization and blastocyst formation. Blastocysts obtained from AgNPs-treated spermatozoa showed lower expression of trophectoderm-associated and pluripotent marker genes. Overall, we propose that AgNPs internalization into spermatozoa may alter sperm physiology, leading to poor fertilization and embryonic development. Such AgNPs-induced reprotoxicity may be a valuable tool as models for testing the safety and applicability of medical devices using AgNPs.

No MeSH data available.


Related in: MedlinePlus