Yi et al.
Journal of Analytical Science and Technology (2023) 14:7
https://doi.org/10.1186/s40543-022-00366-x
RESEARCH ARTICLE
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Open Access
Journal of Analytical Science
and Technology
Inactivation of mammalian spermatozoa
on the exposure of ­TiO2 nanorods deposited
with noble metals
Young‑Joo Yi1*†
   , Love Kumar Dhandole2†
   , Dong‑Won Seo3
   , Sang‑Myeong Lee4
    and Jum Suk Jang2*
    
Abstract 
Titanium dioxide ­(TiO2) nanorods (NRs) are well-known semiconducting and catalytic material that has been widely
applied, but their toxicities have also attracted recent interest. In this study, we investigated and compared the toxic
effects of ­TiO2 NRs and ­TiO2 NRs loaded with Ag or Au NPs on boar spermatozoa. As a result, sperm incubated with
Ag-TiO2 NRs showed lower motility than sperm incubated with controls (with or without ­TiO2 NRs) or Au-TiO2 NRs. In
addition, sperm viability and acrosomal integrity were defective in the presence of Ag-TiO2 NRs, and the generation
of intracellular reactive oxygen species (ROS) increased significantly when spermatozoa were incubated with 20 μg/
ml Ag-TiO2 NRs. We discussed in depth the charge transfer mechanism between enzymatic NADPH and Ag-TiO2 NRs
in the context of ROS generation in spermatozoa. The effects we observed reflected the fertilization competence of
sperm incubated with Ag-TiO2 NRs; specifically sperm penetration and embryonic development rates by in vitro fer‑
tilization were reduced by Ag-TiO2 NRs. To summarize, our findings indicate that exposure to Ag-TiO2 NRs could affect
male fertilization fecundity and caution that care be exercised when using these NRs.
Keywords TiO2 nanorods, Noble metals, In vitro fertilization, NADPH, Embryo development, Au, Ag, Spermatozoa
Introduction
Titanium dioxide nanoparticles ­(TiO2 NPs) are wellknown
semiconducting and catalytic material and have
been widely used for a variety of applications because
of their chemical inertness, thermal–physical stability,
low cost, and environmental friendliness (Wu
et al. 2015). ­TiO2 NPs have shown effective antibacterial
activities (Cornish et  al. 2000; Sichel et  al. 2007;
Dhandole et  al. 2017, 2018). In particular, incorporation
of noble metal (e.g., Ag, Au, or Pt) nanoparticles
on the surface of ­TiO2 particles displayed efficient catalytic
properties and antibacterial activities (Rupa et al.
2007; Traversa et al. 2000). Zhang et al. (2016) synthesized
hetero-nanostructure Au/TiO2 nanocomposites
has good antibacterial activity against Escherichia coli
(1 mg photo-catalyst/ml E. coli suspension) under visible
light and specifically in the dark. Li et al. (2014) proposed
an antibacterial mechanism for plasmonic gold
NP-modified ­TiO2 nanotubes (foil based) in the dark
and found that localized surface plasmon resonance
(LSPR) of gold nanoparticles disrupted electron transfer
in the membrane respiratory system and caused
bacterial death. It hypothesizes that the respiratory
†
Young-Joo Yi and Love Kumar Dhandole have contributed equally to this
work.
*Correspondence:
Young‑Joo Yi
yiyj@scnu.ac.kr
Jum Suk Jang
Jangjs75@jbnu.ac.kr
1
Department of Agricultural Education, College of Education, Sunchon
National University, 255 Jungang‑Ro, Suncheon 57922, Republic of Korea
2
Division of Biotechnology, College of Environmental and Bioresource
Sciences, Jeonbuk National University, 79 Gobong‑Ro, Iksan 54596,
Jeonbuk, Republic of Korea
3
Department of Vaccine Development, Gyeongbuk Institute for Bio
Industry, Andong 36618, Republic of Korea
4
Laboratory of Veterinary Virology, College of Veterinary Medicine,
Chungbuk National University, Cheongju 28644, Republic of Korea
Page 2 of 14Yi et al. Journal of Analytical Science and Technology (2023) 14:7
proteins of microbial membranes may behave as n-type
semiconductors. The physical contact of microbes with
Au nanoparticles will result in Schottky barrier formation
and Fermi level alignment which result in the facile
electrons transfer from microbial membranes to Au
nanoparticles and the resultant increase in surface electron
density of Au nanoparticles. It assumes that the
energy loss may be converted into light energy here and
if the light energy is large enough, it can be absorbed
to induce the LSPR of Au nanoparticles. The plasmonic
hot electrons will flow to the ­TiO2 conduction band
and subsequently to the valence band. Thus, the bacterial
membrane (work as electron donor) steadily loses
electrons in this way and suffers from the reactive oxygen
species (ROS)-independent oxidative stress, which
finally damages the membrane integrity and induces
the cell death.
In addition to its antibacterial activity, recent studies
have reported the effects of ­TiO2 NPs including inflammation,
apoptosis, reactive oxygen species (ROS) production,
and changes in enzyme activity in living cells,
and accumulations in organs (Chang et al. 2013; Shi et al.
2013; Czajka et al. 2015). In an examination of the reproductive
system, intraperitoneal injection of ­TiO2 NPs
affected testis and epididymis in male mice by reducing
sperm counts and motility and increasing sperm abnormalities
and germ cell apoptosis rates, while effects on
livers and kidneys were slight (Guo et  al. 2009). When
­TiO2 NPs were administered to female mice over a long
time, investigators observed ovarian injury, subfertility,
and a low pregnancy rate (Gao et al. 2012), and buffalo
spermatozoa treated with ­TiO2 NPs exhibited DNA
damage and excessive ROS production (Pawar and
Kaul 2014). The potential toxic effects of silver (Ag) and
gold (Au) NPs on reproduction relevant cells have been
observed to be toxic to spermatozoa in a concentration,
size, or dose-dependent manners and have also attracted
research attention (Taylor et al. 2015). Incorporating Ag
NPs (0.1, 1, 10, and 50 μg/ml) into spermatozoa induced
oxidative stress that impaired fertilization and embryonic
development of mouse (Yoisungnern et  al. 2015).
Another study reported that Au NPs reduced sperm
motility, and gold particles can penetrate sperm cells,
which resulted in fragmentation (Wiwanitkit et al. 2009),
while human sperm cultured with Ag NPs at high doses
(greater than 250  μM concentrations) showed slightly
higher cytotoxicity than Au NPs (Moretti et al. 2013).
In this study, we examined the effects of synthesized
crystalline metal oxide ­TiO2 nanorods (NRs) and ­TiO2
NRs loaded with Ag or Au NPs on boar spermatozoa.
In  vitro fertilization (IVF) using pig oocytes matured
in vitro was used to examine the fertilization competence
of spermatozoa incubated without NRs, with ­TiO2 NRs,
Ag-TiO2 NRs, or Au-TiO2 NRs by assessing subsequent
embryonic development. In addition, we investigated the
sperm toxicity mechanism with the help of nicotinamide
adenine dinucleotide phosphate (NADPH), which is used
as an electron donor, and hypothesized a charge transfer
mechanism where living cells lose electrons related to
intracellular ROS generation that eventually compromise
membrane integrity and led to DNA fragmentation.
Materials and methods
Chemical reagents
Commercially available ­TiO2 nanopowder (P25, Degussa)
of average particle size ~ 21  nm was used as the starting
material. ­Na2HPO4 and NaCl were purchased from
Kanto Chemicals (Tokyo, Japan) and Junsei (Kyoto,
Japan), respectively. Noble metal precursor silver nitrate
and gold (III) chloride trihydrate were purchased from
Samchun Chemicals (Seoul, Korea) and Sigma-Aldrich
(St. Louis, MO, USA), respectively.
Unless otherwise noted, all other reagents used in this
study were purchased from Sigma-Aldrich.
Preparation of ­TiO2 nanorods
The procedure used for synthesizing ­TiO2 NRs was documented
in our previous study (Dhandole et  al. 2017).
Briefly, ­TiO2 NRs were synthesized using a molten salt
flux method. In a typical procedure, commercially available
­TiO2 nanopowder (Degussa), NaCl, and ­Na2HPO4
were ground together in the ratio 1:4:1 (by weight) to
form a homogeneous mixture. This mixture was calcined
inside a box furnace at 825 °C for 8 h, cooled to room
temperature (RT), washed with DI water to remove water
soluble salts, collected by filtration paper (< 5 µm), and
rewashed using the same procedure to remove remaining
sodium ions. The collected filtrate was dried overnight at
80 °C inside a hot air oven and then finely ground in a
mortar.
Syntheses of noble metal‑loaded ­TiO2 NRs
We prepared noble metal-loaded (Ag or Au) ­TiO2 NRs by
photo-deposition under a xenon arc lamp (Abet, Japan)
at 150 W. Photo-deposition experiments were performed
in a Pyrex vessel at atmospheric pressure and ambient
temperature. In a typical experiment, we prepared ­TiO2
NR suspensions by dispersing 100 mg of ­TiO2 NR powder
in 60 ml of DI water and then adding 10 ml of methyl
alcohol. The reactor mixture was ultra-sonicated and
stirred for 5 min, and then, 1 wt% aqueous noble metal
precursor solution was added dropwise. Methyl alcohol
scavenged holes and accelerated the rate of metal reduction
during photo-deposition. Aqueous noble metal
solutions were prepared by dissolving noble metal precursors
in 15  g of DI water in vials and then kept in a
Page 3 of 14Yi et al. Journal of Analytical Science and Technology (2023) 14:7	
cool dark place. These solutions were added to ­TiO2 NR
suspensions at a noble metal to ­TiO2 NR weight ratio of
1%, and the reactor suspensions obtained were continuously
stirred for 30 min in the dark and then irradiated
for 60 min under solar light. The colored precipitates that
formed (purple for Au and gray for Ag) were collected by
vacuum filtration on filter paper (0.45 µm), washed with
DI water, dried at 80 °C overnight before catalyst experiments
and characterizations.
Characterizations of noble metal‑loaded ­TiO2 NR catalysts
We performed X-ray diffraction (XRD) structural analysis
using a PANalytical X’pert Pro MPD diffractometer
equipped with a Cu–Kα radiation source (wavelength
Kα1 = 1.540598  Å and Kα2 = 1.544426  Å) operated at
40 kV and 30 mA and a scan rate of 0.03° 2θ ­s−1
over a 2θ
range of 5°–80°. Field emission scanning electron microscopy
(FESEM) was performed using a SUPRA 40VP
unit (Carl Zeiss, Germany) equipped with X-ray energydispersive
spectrometry (EDS). Transmission electron
microscopy (TEM; Jeol JEM-3100F, Tokyo, Japan, at
200 kV) was performed by placing a drop of a sample suspension
in ethanol on a standard carbon-coated copper
grid. X-ray photoelectron spectroscopy (XPS, Thermo
Fisher Scientific, Waltham, MA, USA) using a monochromatic
Al–Kα X-ray source (hν = 1486.6 eV) was used
for elemental quantification and to study valence states.
Catalytic degradation experiment
Degradation experiments were performed in a Pyrex vessel
at atmospheric pressure and ambient temperature
using orange II sodium salt dye and UV–Vis spectrophotometry
(Shimadzu UV-2600 UV–Vis-spectrophotometer)
at the maximum dye absorbance wavelength (λmax;
484  nm). Briefly, commercial NADPH was mixed with
10 μl aqueous orange II sodium salt dye (pH 7.0) under
continuous magnetic stirring. Dye degradation efficiencies
were calculated using the following equation:
where A0 is initial absorbance of the dye solution and At
is dye absorbance during reaction at time t.
Boar sperm preparation and sperm incubation
Liquid boar semen was purchased from a local artificial
insemination (AI) center. The diluted semen was stored
in a storage unit at 17  °C for 5  days. Stock solutions
(1 mg/ml) of ­TiO2 NRs, Ag-TiO2 NRs, and Au-TiO2 NRs
were prepared by suspension in phosphate-buffered solution
(PBS) and sonicated for 10 s at 60 Hz (Daihan Scientific,
Korea) before use. For the incubation experiments,
(1)
Dye degradation efficiency (%) = 1 −
At
A0
× 100
sperm were incubated in Beltsville thawing solution
(BTS; Pursel and Johnson 1976) in the absence or presence
of ­TiO2 NRs (controls), Au-TiO2 NRs, or Ag-TiO2
NRs at final concentrations of 10 or 20 μg/ml, which did
not interfere with sperm movement or fertilization, for
2 h at 37.5 °C. All experiments were repeated at least six
times.
Assessment of sperm motility
Sperm motility was quantified using a computer-assisted
sperm analysis system (CASA, Sperm Class Analyzer®
,
Microptic, Barcelona, Spain). Briefly, a sperm sample
(2  μl) was placed in a pre-warmed (38  °C) Leja counting
slide (Leja Products B.V., Nieuw-Vennep, The Netherlands),
and 10 fields were analyzed at 38 °C to assess
a minimum of 1000 spermatozoa per sample for total
motile sperm (%) and progressive motile sperm (%).
Sperm viability, acrosomal integrity, and intracellular ROS
levels
Sperm cells (1 × ­108
/ml) incubated for 2 h at 37.5 °C were
washed twice with phosphate-buffered saline (PBS) containing
0.1% (w/v) polyvinyl alcohol (PBS-PVA). Sperm
viability was assayed using a LIVE/DEAD®
sperm viability
kit (Molecular Probes, Eugene, OR, USA), which
contained the DNA dyes SYBR14 (final conc. 100  nM)
and propidium iodide (PI; final conc. 10  μM), according
to the manufacturer’s instructions. To assess acrosomal
integrity, sperm were stained with 10 μg/ml lectin
peanut agglutinin-FITC conjugate (PNA) and PI, and
images were then acquired using a fluorescence microscope
(Eclipse Ci, Nikon Instruments Inc., Seoul, Korea)
equipped with a camera (DS-Fi2, Nikon) and imaging
software (version 4.30, Nikon). Spermatozoa were classified
as viable (SYBR14 stained), dead (PI stained), or as
intact (PNA+) or damaged (PNA−) acrosomal sperm.
Intracellular ROS levels were assessed using 1  μM carboxy-DCFDA
(Invitrogen, Eugene, OR, USA), and fluorescence
intensities were measured using a multimode
microplate reader (Spark™
10  M, Tekan, Männedorf,
Switzerland) at excitation (ex.) and emission (em.) wavelengths
of 485 and 520 nm, respectively.
Collection and in vitro maturation (IVM) of pig oocytes
Ovaries were collected from prepubertal gilts at a local
slaughterhouse. Cumulus–oocyte complexes (COCs)
were aspirated from antral follicles (3–6  mm in diameter),
washed three times in HEPES-buffered Tyrode
lactate (TL-HEPES-PVA) medium supplemented with
0.01% (w/v) PVA, and then washed three times with
oocyte maturation medium (Abeydeera et  al. 1998). A
total of 50 COCs were transferred to 500 µl of maturation
medium and layered with mineral oil in a 4-well
Page 4 of 14Yi et al. Journal of Analytical Science and Technology (2023) 14:7
multi-dish equilibrated at 38.5 °C in 5% ­CO2 in air. The
oocyte maturation medium used was tissue culture
medium (TCM) 199 supplemented with 0.1% PVA,
3.05 mM D-glucose, 0.91 mM sodium pyruvate, 0.57 mM
cysteine, 0.5  µg/ml luteinizing hormone, 0.5  µg/ml follicle-stimulating
hormone, 10  ng/ml epidermal growth
factor, 75 µg/ml penicillin G, and 50 µg/ml streptomycin.
Oocytes were cultured in TCM199 for 44 h at 38.5 °C, 5%
­CO2 in air.
In vitro fertilization (IVF) and culture (IVC) of pig oocytes
After IVM, cumulus cells were removed by treating them
with 0.1% hyaluronidase in TL-HEPES-PVA medium
(Abeydeera et al. 1998). Oocytes were then placed into
four 100  μl drops of modified Tris-buffered medium
(mTBM) in a 35-mm polystyrene culture dish and covered
with mineral oil. Spermatozoa were incubated in
BTS in the absence (W/O) or presence of ­TiO2 NRs (controls),
Au-TiO2 NRs, or Ag-TiO2 NRs (final conc.: 10 or
20 μg/ml) for 2 h at 38 °C and washed twice in PBS containing
0.1% PVA (PBS-PVA) at 800× g for 5 min. At the
end of the washing procedure, sperm were resuspended
in mTBM, appropriately diluted, and sperm suspensions
(1 µl) were added to medium containing oocytes to a final
sperm concentration of 1 × ­105
spermatozoa/ml. Oocytes
were co-incubated with spermatozoa for 5 h at 38.5 °C
in a 5% ­CO2 atmosphere. After IVF, oocytes were transferred
to 500 μl porcine zygote medium (PZM-3; Yoshioka
et al. 2002), supplemented with 0.4% bovine serum
albumin, and cultured for an additional 20, 48, or 144 h.
The IVM, IVF, and IVC studies were repeated five times
for each treatment regimen.
Fluorescence staining of oocytes and spermatozoa
Oocytes/embryos were fixed with 2% formaldehyde for
40  min at room temperature (RT), washed twice with
PBS, permeabilized with PBS-Triton X-100 for 30  min,
and stained with 2.5  mg/ml 4′,6-diamidino-2-phenylindole
(DAPI; DNA staining; Molecular Probes, Eugene,
OR, USA) for 40 min. The fertilization statuses of zygotes
(unfertilized, fertilized-monospermic, or fertilized-polyspermic),
cleaved embryo numbers, blastocyst formation,
cell number per blastocyst were determined under
a fluorescence microscope (Nikon Eclipse Ci microscope;
Nikon Instruments Inc., Seoul, Korea). To observe
the attachment of ­TiO2 NRs to spermatozoa, ­TiO2 NRs
(1 mg/ml) were mixed with 1 mM alizarin red S (ARS)
and stored at 4  °C until required (Thurn et  al. 2009).
Spermatozoa (1 × ­108
/ml) were incubated in BTS in the
presence of ­TiO2 NRs-ARS for 2 h at 38.5 °C. Spermatozoa
were then fixed with 2% formaldehyde for 40 min at
RT, washed with PBS three times, stained with 2.5 μg/ml
DAPI and 10 μg/ml PNA for 40 min, and then observed
under a fluorescence microscope (Nikon).
Observations of sperm incubated with ­TiO2 NRs by TEM
Spermatozoa incubated with ­TiO2 NRs, Ag-TiO2 NRs,
or Au-TiO2 NRs were fixed in modified Karnovsky’s fixative
(2% paraformaldehyde and 2% glutaraldehyde in
0.05 M sodium cacodylate buffer (pH 7.2) at 4 °C overnight,
washed three times with 0.05 M sodium cacodylate
buffer at 4 °C for 10 min, and postfixed in 1% osmium
tetroxide in 0.05 M sodium cacodylate buffer for 90 min.
Fixed cells were washed twice with DI water at RT and
stained using 0.5% uranyl acetate at 4 °C overnight. Further,
fixed cells were dehydrated using an increasing ethanol
series (30, 40, 50, 70, 80, 90, and 100%), embedded in
EMbed 812 resin mixture containing DDSA, NMA, and
DMP-30, polymerized at 60  °C for 48  h, and ultra-thin
sections were stained with 2% uranyl acetate for 45 min
followed by lead citrate for 3 min. TEM was conducted
using a Hitachi H-7650 unit at 80 kV (Tokyo, Japan).
Statistical analysis
All experimental data were expressed as mean ± standard
error of the mean (SEM), and analyzed using one-way
ANOVA in GraphPad PRISM®
(GraphPad software, San
Diego, CA, USA). The completely randomized design
was applied, and Tukey’s multiple comparison test was
performed to compare values of individual treatments.
Results are considered statistically significant at *p < 0.05,
**p < 0.01 and ***p < 0.001.
Results
Characterizations of noble metal‑loaded ­TiO2 NRs
The XRD patterns of ­TiO2 NRs are shown in Fig.  1A.
Major diffraction peaks at 2θ = 27.5, 36.1, and 54.4° correspond
to (110), (101), and (211) crystal planes, which
is the most reported phase of rutile (JCPDS 89-4202)
(Dhandole et  al. 2016). However, small length NRs (or
broken residuals) were also observed which contributed
small portion in the overall synthesized product. The
elemental analysis EDS is shown in Fig. 1E–H and represents
the elemental quantifications and noble metals (1
wt%) observed on the surface of ­TiO2 NRs. Particle size
distribution histogram for ­TiO2 NR and deposited noble
metals (Au and Ag) were illustrated in Fig. 1I, J. Ag and
Au particle size was determined by TEM analysis.
As shown in Fig.  2A (a, b), Ag and Au NPs were
observed on the surfaces of ­TiO2 NRs and both had a
diameter of 20–30 nm as determined by TEM. The oxidation
states of elements and the chemical compositions
of the as-synthesized materials were determined by XPS.
Figure 2B shows the high-resolution XPS spectra for Ti
Page 5 of 14Yi et al. Journal of Analytical Science and Technology (2023) 14:7	
Fig. 1  Characteristics of novel metal-loaded ­TiO2 NRs. A XRD image of rutile ­TiO2 NRs. FESEM images of B bare ­TiO2 NRs, C Ag-loaded ­TiO2 NRs, and
D Au-loaded ­TiO2 NRs. EDS spectrum and elemental quantification table (inset) of E ­TiO2-NRs, F 2-time washed ­TiO2-NRs, G Au (1wt%)-TiO2-NRs, and
H Ag (1wt%)-TiO2-NRs. Particle size (length) distribution histogram for I ­TiO2 NR, and J deposited noble metals (Au and Ag)
Page 6 of 14Yi et al. Journal of Analytical Science and Technology (2023) 14:7
Fig. 2  Nanoparticle attachment to ­TiO2 NRs as determined by electron microscopy. A TEM images of (a) silver (Ag) nanoparticle-loaded ­TiO2
NRs and (b) gold (Au) nanoparticle-loaded ­TiO2 NRs. Dotted circles show Ag and Au nanoparticles on the surfaces of ­TiO2 NRs. (c) and (d) show
respective TEM maps. B High-resolution XPS spectra of Ti 2p, O 1s, Ag 3d, and Au 4f oxidation peaks of (a) bare ­TiO2 NRs, (b) Ag-loaded ­TiO2 NRs, and
(c) Au-loaded ­TiO2 NRs
Page 7 of 14Yi et al. Journal of Analytical Science and Technology (2023) 14:7	
2p and O 1s of ­TiO2 NRs and the noble metal-loaded
­TiO2 NRs. XPS peaks at around 458.5 and 464.0 eV correspond
to Ti ­2p3/2 and Ti ­2p1/2 spin–orbit pairs, respectively,
confirming that titanium doublet peaks were due
to the Ti (IV) oxidation state (Li et al. 2005; Ohno et al.
2003). The peak at ~ 529 eV was ascribed to oxygen. The
shifts of Ag and Au peaks to lower energies compared
with bulk material confirmed large quantities of both
metals deposited on the surfaces of ­TiO2 NRs and that
strong metallic interactions had formed between noble
metal NPs and the ­TiO2 NRs. The XPS spectra of Ag
3d and Au 4f contained doublet peaks located at ~ 367.7
and ~ 373.5 eV, which corresponded to the reported binding
energies of Ag ­3d5/2 and Ag ­3d3/2, respectively, and
peaks at ~ 83.0 and ~ 87.05  eV, which corresponded to
the binding energies of Au ­4f7/2 and Au ­4f5/2, respectively
(Su et al. 2012; Zhang et al. 2014; Haruta 1997; Ma et al.
2014).
Sperm motility in the presence of noble metal‑loaded ­TiO2
NRs
We assessed sperm motilities using a CASA system;
motility parameters after treatments for 10 min and 2 h
are provided in Fig.  3. The percentage of total motile
sperm after 10 min of incubation was higher for sperm
treated with 10  μg/ml of Au-TiO2 NRs than for sperm
treated with 20 μg/ml of Ag-TiO2 NRs; however, differences
between treatment groups were not significant
(Fig. 3A, B). After incubation for 2 h, motility was significantly
greater for non-treated controls than in the other
groups, and the motility of sperm incubated with 20 μg/
ml of Ag-TiO2 NRs was significantly less than in the other
groups (*p < 0.05, **p < 0.01 and ***p < 0.001; Fig. 3C). The
percentage of progressive motile spermatozoa was higher
after treatment with 10 μg/ml Au-TiO2 NRs for 10 min,
but then decreased after treatment for 2 h. In particular,
we observed significant progressive loss of motility after
treatment with 20  μg/ml Ag-TiO2 NRs (**p < 0.01 and
***p < 0.001; Fig. 3D). Motility refers to the ability of spermatozoa
to move and swim independently in the female
reproductive tract and is essential for successful fertilization.
These results suggest that Ag-TiO2 NRs affects
sperm movement either physically or chemically.
Sperm viability, acrosomal integrity, and ROS levels
in spermatozoa incubated with ­TiO2 NRs
Figure 4 shows viability and intact acrosome results for
sperm incubated for 30 min or 2 h with ­TiO2 NRs, AgTiO2
NRs, or Au-TiO2 NRs. Percentages of viable sperm
were higher after treatment with 10 μg/ml Au-TiO2 NRs
and for controls than after treatment with 20 μg/ml AuTiO2
NRs 10 or 20 μg/ml Ag-TiO2 NRs (75.7–76.3% vs.
63.0–73.6%; p < 0.05, p < 0.01 and p < 0.001; Fig.  4A).
These results indicate that Ag-TiO2 NRs at 10 and 20 μg/
ml adversely affected the viability of spermatozoa, and
the higher decrement obtained for 2  h incubation signifies
that sperm movement might be disturbed under
high doses of Ag-TiO2 NRs (p < 0.05 and p < 0.01; Fig. 4B).
Regarding acrosomal integrity, there was a high percentage
of intact acrosome spermatozoa (PNA-/PI-) in the no
treatment sample, but damaged acrosomes significantly
increased in the 20 μg/ml Ag-TiO2 NR group after incubation
of 30 min or 2 h than in the other groups (p < 0.05,
p < 0.01 and p < 0.001; Fig.  4C, D). To understand the
effects of catalysts on the motility and viability of spermatozoa,
we measured intracellular ROS levels after treating
sperm for 2 h. Interestingly, ROS levels were found
to be significantly higher after treatment with Ag-TiO2
NRs than for the other treatments (p < 0.05 and p < 0.001;
Fig. 4E).
Spermatozoa were incubated with ARS-coated ­TiO2
NRs (controls, Ag-loaded, and Au-loaded) for 4  h,
washed twice with PBS-PVA, fixed, and stained with
PNA (sperm acrosome, green) and DAPI (DNA, blue;
A–D); all NRs showed sufficient ARS (red fluorescence;
Fig. 5). Scattered or clumped coated ­TiO2 NRs (stained
red) were attached to sperm heads, midsections, or tails
(white arrows) after each treatment (Fig. 5A–D). In TEM
analysis, Ag-TiO2 NRs and nanoparticles were observed
in acrosomal membrane (white arrows; Fig. 5E) (Lan and
Yang 2012; Lafuente et al. 2016; Li et al. 2005, 2014; Ma
et al. 2014). ­TiO2 NRs attached to spermatozoa were also
observed during IVF, which presumably would prevent
sperm penetrating oocytes (Fig. 5F).
Fertilization and embryo development rates on sperm
incubated with ­TiO2 NRs
To determine fertilization rates, oocytes were inseminated
with spermatozoa incubated with controls and ­TiO2
NRs, Au-TiO2 NRs, or Ag-TiO2 NRs for 2 h (Fig. 6A). The
total fertilization rate including the rate of monospermic
and polyspermic oocytes was lower when spermatozoa
were incubated with 20  μg/ml Ag-TiO2 NRs (monospermic:
33.9%, polyspermy: 0%) or 20  μg/ml Au-TiO2 NRs
(monospermic: 33.4%, polyspermy: 0%) than controls
(monospermic: 46.9–64.4%, polyspermy: 12.4–16.7%)
and other treatments (monospermic: 41.5–54.8%, polyspermy:
0–15.8%), but there were no significantly differences
among groups (Fig.  6A). As regards embryonic
development, we observed more cleaved oocytes from
IVF performed using control sperm (83.1%) and a significantly
lower cleavage rate when sperm were incubated
with 20 μg/ml Ag-TiO2 NRs (54.1%) or 20 μg/ml Au-TiO2
NRs (55.6%) than in the other groups (60.6–75.1%, p < 0.05;
Fig.  6B). Furthermore, the blastocyst formation rate was
higher in the control group (27.1%) than in the other
Page 8 of 14Yi et al. Journal of Analytical Science and Technology (2023) 14:7
groups (10.2–4.1%; Fig. 6C), and when oocytes were fertilized
with spermatozoa incubated with 20 μg/ml Ag-loaded
­TiO2 NRs, the lowest blastocyst rates were observed (2.2%,
p < 0.05 and p < 0.001; Fig. 6C). However, mean cell number
per blastocyst in the groups were not significantly different
(30.1–45.0 cells/blastocyst).
Intracellular ROS generation by Ag‑TiO2 NRs
in spermatozoa
To investigate intracellular ROS generation in sperm
exposed to Ag-TiO2 NR, we first examined the role of
NADPH during the process of electron donation. Specifically,
we tested the effects of NADPH activity over
the degradation of organic orange II dye in the presence
of Ag-TiO2 NR catalyst. Orange (II) dye contains an azo
linkage, which forms N=N bonds with its chromophores
benzene and naphthalene, and this linkage is sensitive
to active radical species such as OH·, HOO·, and O·2
−
(Dhandole et al. 2017). We observed low dye concentrations
in the presence of 0.05–2 mM NADPH and 0.5 mg/
ml Ag-TiO2 NRs (Fig.  7A), but single treatments with
NADPH or Ag-loaded ­TiO2 NRs had no effect. This result
indicates that the synergistic effect of NADPH and AgTiO2
NR treatment for 4 h increased the dye degradation
rate as increasing concentration of NADPH (Fig.  7A).
Based on this result, we fixed the concentration of
Fig. 3  Comparison of total motile sperm and progressive motile sperm after incubation with or without (W/O) ­TiO2 NRs. Boar spermatozoa were
incubated in the presence of ­TiO2 NRs for 10 min (A, B) and 2 h (C, D). Values are expressed as mean ± SEM. Lines (or dotted lines) among columns
denote significant differences at *p < 0.05, **p < 0.01 and ***p < 0.001
Page 9 of 14Yi et al. Journal of Analytical Science and Technology (2023) 14:7	
Fig. 4  Assessment of sperm viability and acrosomal integrity. Boar spermatozoa were incubated in the absence (W/O) or presence of NRs. Live
(viable) spermatozoa were counted after incubation for 30 min (A) or 2 h (B). Also, intact acrosomes were examined using PNA and PI staining after
incubation for 30 min (C) or 2 h (D). E Intracellular ROS generation in spermatozoa exposed to ­TiO2 NRs. Fluorescence intensities were measured
in sperm stained with carboxy-DCFDA. Experiments were repeated five times with spermatozoa from two different boars. Values are expressed as
mean ± SEM. Lines (or dotted lines) among columns denote significant differences at *p < 0.05, **p < 0.01, and ***p < 0.001
Fig. 5  Spermatozoa were incubated with alizarin red S (ARS, red), and stained with PNA (sperm acrosome, green) and DAPI (DNA, blue). A Control
(without [W/O] catalyst), B ­TiO2 NRs, C Ag-loaded ­TiO2 NRs, and D Au-loaded ­TiO2 NRs for 4 h. E TEM image of ­TiO2 NRs attached to sperm acrosome
and nuclear membrane (white arrows). F ­TiO2 NRs attached to spermatozoa inhibited sperm penetration during IVF
Page 10 of 14Yi et al. Journal of Analytical Science and Technology (2023) 14:7
NADPH at 0.2 mM and conducted the same experiment
but replaced the organic dye with fresh sperm (Fig. 7B).
We observed higher ROS levels when NADPH (0.2 mM)
and Ag-loaded ­TiO2 NRs were treated in combination;
ROS production was low when only NADPH was administered
(Fig.  7B). These results indicate the importance
of NADPH as an electron donor (Aitken et al. 1997). In
the presence of low NADPH levels, Ag-loaded ­TiO2 NRs
show enhanced generating of ROS, which suggests these
NRs acted as charge carriers from NADPH donor ligands
(Fig. 8).
Discussion
Remarkable advances in nanotechnology have made
many novel biomedical applications possible, especially
in the reproductive biology field. However, due to
the environmental effects of the widespread use of NPs
and their adverse effects on animal germ cells, their use
should be subjected to strict review (Falchi et al. 2018).
In particular, NPs have been recently reported to be toxic
to male reproductive organs and germ cells. Small NPs
easily penetrate cell membranes, such as the blood–testis
barrier, accumulate or deposit in testis, and disrupt sperm
formation and development (Lan and Yang 2012). In rats
Fig. 6  Fertilization competence of sperm incubated with/without ­TiO2 NRs. A Oocytes were fertilized with sperm incubated with control (W/O),
Ag-loaded, and Au-loaded ­TiO2 NRs. B After IVF, fertilized oocytes were cultured for 144 h to observe subsequent embryo development. Numbers
of inseminated oocytes are indicated in parentheses. Experiments were independently performed five times. Values are expressed as mean ± SEM.
Lines (or dotted lines) among columns denote significant differences at *p < 0.05 and **p < 0.01
Page 11 of 14Yi et al. Journal of Analytical Science and Technology (2023) 14:7	
Fig. 7  ROS generation in spermatozoa incubated with Ag-loaded ­TiO2 NRs using NADPH. A Dye degradation of Ag-loaded ­TiO2 NRs in the presence
of NADPH. B ROS generation (in sperm) incubated with different catalysts Ag nanoparticles, ­TiO2 NRs, and Ag-TiO2 NR in the presence of commercial
NADPH. Values are expressed as mean ± SEM. Lines (or dotted lines) among columns denote significant differences at *p < 0.05 and **p < 0.01
Fig. 8  Schematic representation of electron transfer mechanism in boar spermatozoa incubated with Ag-loaded ­TiO2 NR catalyst
Page 12 of 14Yi et al. Journal of Analytical Science and Technology (2023) 14:7
fed Ag NPs, sex hormone levels decreased and abnormal
sperm morphology and motility increased (Neill et  al.
2009; Baki et al. 2014; Lafuente et al. 2016), and administering
Au NPs to mice caused abnormal sperm chromatin
remodeling and DNA damage (Nazar et al. 2016). In
similar experiments, functional defects and DNA damage
in spermatozoa were observed in male mice administered
­TiO2 NPs (Smith et al. 2015), and direct exposure to NPs
interfered with sperm function, for example, bull sperm
exposed to Au NPs showed impaired motility and fertility
(Taylor et  al. 2014), and human sperm treated with
44 ppm of Au NPs showed 25% lower motility than nontreated
controls (Wiwanitkit et al. 2009). In the present
study, direct exposure of Ag-loaded ­TiO2 NRs to boar
spermatozoa decreased motility, viability, and acrosomal
integrity, which resulted in lower fertilization and embryonic
development rates.
Spermatozoa are highly sensitive cells dedicated to fertilizing
oocytes, and many factors can influence sperm
attachment and fertilization. The most problematic factor
is the intracellular production of ROS and related oxidative
stress, which adversely affects sperm survival and
fertility (Saadeldin et al. 2020). Vernet et al. (2001) suggested
that intracellular ROS generation might be a collective
effect of the mitochondrial respiratory chain and
NADPH oxidase system in sperm plasma membranes.
The mitochondrial electron transport chain reaction produces
ROS under physiological conditions, and this is
correlated with physical activity (Riaz et al. 2017; Vernet
et al. 2004). However, recent studies have confirmed that
the cytotoxic effect of Ag NPs can also induce intracellular
ROS production, which is responsible for most of
the common abnormalities in spermatozoa, such as disrupted
chromatin, bent tails, curved midsections, DNA
fragmentation, mitochondrial damage, respiratory chain
disruption, oxidative stress, and chromosomal aberrations
(Haase et al. 2012; Beer et al. 2012; Wang et al.
2017).
Based on a literature review, we report an electron
transfer mechanism that underlies spermatozoa inactivation
and present for the first time the effect metal oxide
nanoparticles have on intracellular ROS stress in spermatozoa
caused by mitochondrial transmembrane electron
transport. We present the suggested general electron
transfer mechanism in a schematic diagram in Fig. 8. The
physical contact of sperm cell with Ag-loaded ­TiO2 NR
will result in Schottky barrier whose Fermi-level alignment
(based on band theory) results in the facile electron
transport from spermatozoa to Ag NPs over the surface
of ­TiO2 NRs. The electron donor ligand of Ag NP might
accept the transmembrane electrons of sperm enzymatic
NADPH after mitochondrial electron transport chain
reactions have been disrupted. The affinity of the ­Ag+
metallic state is higher than the donor ligand, and reduction
potential is a parameter of thermodynamic reactions
(Vernet et al. 2001; Prasad et al. 2017). The reduced species,
­Ag0
, transfer electrons to the conduction band of
­TiO2 because the band edge of the ­TiO2 is lower than that
of the Ag-loaded NPs (as shown in Fig.  7; Kochuveedu
et al. 2013). After an electron is released, Ag re-oxidizes,
and the cycle continues until the donor ligand actively
participates in the reaction. Meanwhile, conduction band
electrons reduce oxygen molecules to form OH·, ­O2
−
,
and OOH· radicals (Dhandole et al. 2017), which cause
cell damage and DNA fragmentation. In a recent study, it
was reported that morin and rutin have protective effects
on rat testis from oxidative damage caused by ROS associated
with ­TiO2 NP intake (Hussein et al. 2019). Therefore,
a further study on antioxidants to mitigate the
toxicity and oxidative damage associated with ROS production
derived from Ag- or TiO2 NRs is needed in the
future.
Conclusions
In the present study, Ag or Au NPs were incorporated on
the surfaces of ­TiO2 NRs by photo-deposition, and their
effects on boar spermatozoa were examined. In addition,
porcine oocytes matured in vitro were inseminated
with sperm that had been incubated in the presence of
the noble metal-loaded NRs, and fertilization rates and
embryo developments were investigated to verify germ
cell toxicity in preimplantation embryos. Our experimental
results indicated that Ag-TiO2 NRs at 20  μg/ml
generated ROS that decreased the fertilization rate and
reduced sperm viability and acrosome integrity. Furthermore,
our investigation of the charge transfer mechanism
and ROS generation showed that a catalytic redox reaction
took place in sperm nuclei and that mitochondrial
NADPH functions as an electron donor and activates Agloaded
­TiO2 NRs.
Acknowledgements
Y-JY was supported by National Research Foundation of Korea (NRF) Grants
funded by the Korea government (MSIT) (NRF-2013R1A6A3A04063769 and
NRF-2020R1A2C1014007), and JSJ was supported by research funds of Jeon‑
buk National University in 2022.
Author contributions
Y-JY, and LKD have contributed equally to this work. Y-JY, LKD, and JSJ contrib‑
uted to conceptualization. Y-JY, LKD, D-WS, and S-ML were involved in investi‑
gation. Y-JY and LKD contributed to analysis. Y-JY, LKD, and JSJ were involved
in writing—original draft. Y-JY, LKD, and JSJ contributed to writing—review
and editing. All authors read and approved the final manuscript.
Funding
This work was supported by National Research Foundation of Korea (NRF)
Grants funded by the Korea government (MSIT) (NRF-2013R1A6A3A04063769
and NRF-2020R1A2C1014007).
Page 13 of 14Yi et al. Journal of Analytical Science and Technology (2023) 14:7	
Availability of data and materials
Upon reasonable request, the datasets of this study can be available from the
corresponding author.
Declarations
Ethics approval and consent to participate
This article does not require IRB/IACUC approval because there are no human
and animal participants.
Competing interests
No potential conflict of interest relevant to this article was reported.
Received: 14 September 2022 Accepted: 28 December 2022
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