Differential nanoreprotoxicity of silver nanoparticles in male somatic cells and spermatogonial stem cells.
BACKGROUND
Silver nanoparticles (AgNPs) possess unique physical, chemical, and biological properties. AgNPs have been increasingly used as anticancer, antiangiogenic, and antibacterial agents for the treatment of bacterial infections in open wounds as well as in ointments, bandages, and wound dressings. The present study aimed to investigate the effects of two different sizes of AgNPs (10 nm and 20 nm) in male somatic Leydig (TM3) and Sertoli (TM4) cells and spermatogonial stem cells (SSCs).


METHODS
Here, we demonstrate a green and simple method for the synthesis of AgNPs using Bacillus cereus culture supernatants. The synthesized AgNPs were characterized using ultraviolet and visible absorption spectroscopy, X-ray diffraction, Fourier transform infrared spectroscopy, and transmission electron microscopy (TEM). The toxicity of the synthesized AgNPs was evaluated by the effects on cell viability, metabolic activity, oxidative stress, apoptosis, and expression of genes encoding steroidogenic and tight junction proteins.


RESULTS
AgNPs inhibited the viability and proliferation of TM3 and TM4 cells in a dose- and size-dependent manner by damaging cell membranes and inducing the generation of reactive oxygen species, which in turn affected SSC growth on TM3 and TM4 as feeder cells. Small AgNPs (10 nm) were more cytotoxic than medium-sized nanoparticles (20 nm). TEM revealed the presence of AgNPs in the cell cytoplasm and nucleus, and detected mitochondrial damage and enhanced formation of autosomes and autolysosomes in the AgNP-treated cells. Flow cytometry analysis using Annexin V/propidium iodide staining showed massive cell death by apoptosis or necrosis. Real-time polymerase chain reaction and western blot analyses indicated that in TM3 and TM4 cells, AgNPs activated the p53, p38, and pErk1/2 signaling pathways and significantly downregulated the expression of genes related to testosterone synthesis (TM3) and tight junctions (TM4). Furthermore, the exposure of TM3 and TM4 cells to AgNPs inhibited proliferation and self-renewal of SSCs.


CONCLUSIONS
Our results suggest that AgNPs exhibit size-dependent nanoreprotoxicity in male somatic cells and SSCs, strongly suggesting that applications of AgNPs in commercial products must be carefully evaluated. Further studies of AgNPs-induced nanoreprotoxicity in animal models are required.
Department of Animal Biotechnology, Konkuk University, Seoul, South Korea.
Xi-Feng
XF
Zhang
Department of Animal Biotechnology, Konkuk University, Seoul, South Korea.
Yun-Jung
YJ
Choi
Department of Animal Biotechnology, Konkuk University, Seoul, South Korea.
Jae Woong
JW
Han
Department of Animal Biotechnology, Konkuk University, Seoul, South Korea.
Eunsu
E
Kim
Department of Animal Biotechnology, Konkuk University, Seoul, South Korea.
Jung Hyun
JH
Park
Department of Animal Biotechnology, Konkuk University, Seoul, South Korea.
Sangiliyandi
S
Gurunathan
Department of Animal Biotechnology, Konkuk University, Seoul, South Korea.
Jin-Hoi
JH
Kim
D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D007985:Leydig Cells / Q000187:drug effects*; D008297:Male; D053768:Metal Nanoparticles / Q000737:chemistry / Q000633:toxicity; D051379:Mice; D018384:Oxidative Stress / Q000187:drug effects; D012708:Sertoli Cells / Q000187:drug effects*; D012834:Silver / Q000737:chemistry / Q000633:toxicity
D012834:Silver
Leydig cells; Sertoli cells; apoptosis; oxidative stress; tight junction proteins