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Titanium dioxide ( TiO ) 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 TiO NRs and TiO NRs loaded with Ag or Au NPs on boar spermatozoa. As a result, sperm incubated with 2 2 Ag‑ TiO NRs showed lower motility than sperm incubated with controls (with or without TiO NRs) or Au‑ TiO NRs. In 2 2 2 addition, sperm viability and acrosomal integrity were defective in the presence of Ag‑ TiO NRs, and the generation of intracellular reactive oxygen species (ROS) increased significantly when spermatozoa were incubated with 20 μg/ ml Ag‑ TiO NRs. We discussed in depth the charge transfer mechanism between enzymatic NADPH and Ag‑ TiO NRs 2 2 in the context of ROS generation in spermatozoa. The effects we observed reflected the fertilization competence of sperm incubated with Ag‑ TiO NRs; specifically sperm penetration and embryonic development rates by in vitro fer ‑ tilization were reduced by Ag‑ TiO NRs. To summarize, our findings indicate that exposure to Ag‑ TiO NRs could affect 2 2 male fertilization fecundity and caution that care be exercised when using these NRs. Keywords TiO nanorods, Noble metals, In vitro fertilization, NADPH, Embryo development, Au, Ag, Spermatozoa of their chemical inertness, thermal–physical stabil- Introduction ity, low cost, and environmental friendliness (Wu Titanium dioxide nanoparticles (TiO NPs) are well- et al. 2015). TiO NPs have shown effective antibacte - known semiconducting and catalytic material and have rial activities (Cornish et al. 2000; Sichel et al. 2007; been widely used for a variety of applications because Dhandole et al. 2017, 2018). In particular, incorpora- tion of noble metal (e.g., Ag, Au, or Pt) nanoparticles Young‑ Joo Yi and Love Kumar Dhandole have contributed equally to this on the surface of TiO particles displayed efficient cata - work. lytic properties and antibacterial activities (Rupa et al. *Correspondence: 2007; Traversa et al. 2000). Zhang et al. (2016) synthe- Young‑Joo Yi sized hetero-nanostructure Au/TiO nanocomposites firstname.lastname@example.org has good antibacterial activity against Escherichia coli Jum Suk Jang Jangjs75@jbnu.ac.kr (1 mg photo-catalyst/ml E. coli suspension) under visi- Department of Agricultural Education, College of Education, Sunchon ble light and specifically in the dark. Li et al. (2014) pro - National University, 255 Jungang‑Ro, Suncheon 57922, Republic of Korea posed an antibacterial mechanism for plasmonic gold Division of Biotechnology, College of Environmental and Bioresource Sciences, Jeonbuk National University, 79 Gobong‑Ro, Iksan 54596, NP-modified TiO nanotubes (foil based) in the dark Jeonbuk, Republic of Korea and found that localized surface plasmon resonance Department of Vaccine Development, Gyeongbuk Institute for Bio (LSPR) of gold nanoparticles disrupted electron trans- Industry, Andong 36618, Republic of Korea Laboratory of Veterinary Virology, College of Veterinary Medicine, fer in the membrane respiratory system and caused Chungbuk National University, Cheongju 28644, Republic of Korea bacterial death. It hypothesizes that the respiratory © The Author(s) 2023. 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Journal of Analytical Science and Technology (2023) 14:7 Page 2 of 14 proteins of microbial membranes may behave as n-type Ag-TiO NRs, or Au-TiO NRs by assessing subsequent 2 2 semiconductors. The physical contact of microbes with embryonic development. In addition, we investigated the Au nanoparticles will result in Schottky barrier forma- sperm toxicity mechanism with the help of nicotinamide tion and Fermi level alignment which result in the fac- adenine dinucleotide phosphate (NADPH), which is used ile electrons transfer from microbial membranes to Au as an electron donor, and hypothesized a charge transfer nanoparticles and the resultant increase in surface elec- mechanism where living cells lose electrons related to tron density of Au nanoparticles. It assumes that the intracellular ROS generation that eventually compromise energy loss may be converted into light energy here and membrane integrity and led to DNA fragmentation. if the light energy is large enough, it can be absorbed to induce the LSPR of Au nanoparticles. The plasmonic Materials and methods hot electrons will flow to the TiO conduction band Chemical reagents and subsequently to the valence band. Thus, the bacte - Commercially available TiO nanopowder (P25, Degussa) rial membrane (work as electron donor) steadily loses of average particle size ~ 21 nm was used as the start- electrons in this way and suffers from the reactive oxy - ing material. Na HPO and NaCl were purchased from 2 4 gen species (ROS)-independent oxidative stress, which Kanto Chemicals (Tokyo, Japan) and Junsei (Kyoto, finally damages the membrane integrity and induces Japan), respectively. Noble metal precursor silver nitrate the cell death. and gold (III) chloride trihydrate were purchased from In addition to its antibacterial activity, recent studies Samchun Chemicals (Seoul, Korea) and Sigma-Aldrich have reported the effects of TiO NPs including inflam - (St. Louis, MO, USA), respectively. mation, apoptosis, reactive oxygen species (ROS) pro- Unless otherwise noted, all other reagents used in this duction, and changes in enzyme activity in living cells, study were purchased from Sigma-Aldrich. and accumulations in organs (Chang et al. 2013; Shi et al. 2013; Czajka et al. 2015). In an examination of the repro-Preparation of TiO nanorods ductive system, intraperitoneal injection of TiO NPs The procedure used for synthesizing TiO NRs was doc- 2 2 affected testis and epididymis in male mice by reducing umented in our previous study (Dhandole et al. 2017). sperm counts and motility and increasing sperm abnor-Briefly, TiO NRs were synthesized using a molten salt malities and germ cell apoptosis rates, while effects on flux method. In a typical procedure, commercially avail - livers and kidneys were slight (Guo et al. 2009). When able TiO nanopowder (Degussa), NaCl, and Na HPO 2 2 4 TiO NPs were administered to female mice over a long were ground together in the ratio 1:4:1 (by weight) to time, investigators observed ovarian injury, subfertil- form a homogeneous mixture. This mixture was calcined ity, and a low pregnancy rate (Gao et al. 2012), and buf- inside a box furnace at 825 °C for 8 h, cooled to room falo spermatozoa treated with T iO NPs exhibited DNA temperature (RT), washed with DI water to remove water damage and excessive ROS production (Pawar and soluble salts, collected by filtration paper (< 5 µm), and Kaul 2014). The potential toxic effects of silver (Ag) and rewashed using the same procedure to remove remaining gold (Au) NPs on reproduction relevant cells have been sodium ions. The collected filtrate was dried overnight at observed to be toxic to spermatozoa in a concentration, 80 °C inside a hot air oven and then finely ground in a size, or dose-dependent manners and have also attracted mortar. research attention (Taylor et al. 2015). Incorporating Ag NPs (0.1, 1, 10, and 50 μg/ml) into spermatozoa induced Syntheses of noble metal‑loaded TiO NRs oxidative stress that impaired fertilization and embry- We prepared noble metal-loaded (Ag or Au) TiO NRs by onic development of mouse (Yoisungnern et al. 2015). photo-deposition under a xenon arc lamp (Abet, Japan) Another study reported that Au NPs reduced sperm at 150 W. Photo-deposition experiments were performed motility, and gold particles can penetrate sperm cells, in a Pyrex vessel at atmospheric pressure and ambient which resulted in fragmentation (Wiwanitkit et al. 2009), temperature. In a typical experiment, we prepared T iO while human sperm cultured with Ag NPs at high doses NR suspensions by dispersing 100 mg of TiO NR pow- (greater than 250 μM concentrations) showed slightly der in 60 ml of DI water and then adding 10 ml of methyl higher cytotoxicity than Au NPs (Moretti et al. 2013). alcohol. The reactor mixture was ultra-sonicated and In this study, we examined the effects of synthesized stirred for 5 min, and then, 1 wt% aqueous noble metal crystalline metal oxide TiO nanorods (NRs) and TiO precursor solution was added dropwise. Methyl alcohol 2 2 NRs loaded with Ag or Au NPs on boar spermatozoa. scavenged holes and accelerated the rate of metal reduc- In vitro fertilization (IVF) using pig oocytes matured tion during photo-deposition. Aqueous noble metal in vitro was used to examine the fertilization competence solutions were prepared by dissolving noble metal pre- of spermatozoa incubated without NRs, with TiO NRs, cursors in 15 g of DI water in vials and then kept in a 2 Yi et al. Journal of Analytical Science and Technology (2023) 14:7 Page 3 of 14 cool dark place. These solutions were added to TiO NR sperm were incubated in Beltsville thawing solution suspensions at a noble metal to TiO NR weight ratio of (BTS; Pursel and Johnson 1976) in the absence or pres- 1%, and the reactor suspensions obtained were continu- ence of TiO NRs (controls), Au-TiO NRs, or Ag-TiO 2 2 2 ously stirred for 30 min in the dark and then irradiated NRs at final concentrations of 10 or 20 μg/ml, which did for 60 min under solar light. The colored precipitates that not interfere with sperm movement or fertilization, for formed (purple for Au and gray for Ag) were collected by 2 h at 37.5 °C. All experiments were repeated at least six vacuum filtration on filter paper (0.45 µm), washed with times. DI water, dried at 80 °C overnight before catalyst experi- ments and characterizations. Assessment of sperm motility Sperm motility was quantified using a computer-assisted Characterizations of noble metal‑loaded TiO NR catalysts sperm analysis system (CASA, Sperm Class Analyzer , We performed X-ray diffraction (XRD) structural analy - Microptic, Barcelona, Spain). Briefly, a sperm sample sis using a PANalytical X’pert Pro MPD diffractometer (2 μl) was placed in a pre-warmed (38 °C) Leja count- equipped with a Cu–K radiation source (wavelength ing slide (Leja Products B.V., Nieuw-Vennep, The Neth - K = 1.540598 Å and K = 1.544426 Å) operated at erlands), and 10 fields were analyzed at 38 °C to assess α1 α2 −1 40 kV and 30 mA and a scan rate of 0.03° 2θ s over a 2θ a minimum of 1000 spermatozoa per sample for total range of 5°–80°. Field emission scanning electron micros- motile sperm (%) and progressive motile sperm (%). copy (FESEM) was performed using a SUPRA 40VP unit (Carl Zeiss, Germany) equipped with X-ray energy- Sperm viability, acrosomal integrity, and intracellular ROS dispersive spectrometry (EDS). Transmission electron levels microscopy (TEM; Jeol JEM-3100F, Tokyo, Japan, at Sperm cells (1 × 10 /ml) incubated for 2 h at 37.5 °C were 200 kV) was performed by placing a drop of a sample sus- washed twice with phosphate-buffered saline (PBS) con - pension in ethanol on a standard carbon-coated copper taining 0.1% (w/v) polyvinyl alcohol (PBS-PVA). Sperm grid. X-ray photoelectron spectroscopy (XPS, Thermo viability was assayed using a LIVE/DEAD sperm via- Fisher Scientific, Waltham, MA, USA) using a mono - bility kit (Molecular Probes, Eugene, OR, USA), which chromatic Al–K X-ray source (hν = 1486.6 eV) was used contained the DNA dyes SYBR14 (final conc. 100 nM) for elemental quantification and to study valence states. and propidium iodide (PI; final conc. 10 μM), accord - ing to the manufacturer’s instructions. To assess acro- Catalytic degradation experiment somal integrity, sperm were stained with 10 μg/ml lectin Degradation experiments were performed in a Pyrex ves- peanut agglutinin-FITC conjugate (PNA) and PI, and sel at atmospheric pressure and ambient temperature images were then acquired using a fluorescence micro - using orange II sodium salt dye and UV–Vis spectropho- scope (Eclipse Ci, Nikon Instruments Inc., Seoul, Korea) tometry (Shimadzu UV-2600 UV–Vis-spectrophotom- equipped with a camera (DS-Fi2, Nikon) and imaging eter) at the maximum dye absorbance wavelength (λ ; software (version 4.30, Nikon). Spermatozoa were classi- max 484 nm). Briefly, commercial NADPH was mixed with fied as viable (SYBR14 stained), dead (PI stained), or as 10 μl aqueous orange II sodium salt dye (pH 7.0) under intact (PNA+) or damaged (PNA−) acrosomal sperm. continuous magnetic stirring. Dye degradation efficien - Intracellular ROS levels were assessed using 1 μM car- cies were calculated using the following equation: boxy-DCFDA (Invitrogen, Eugene, OR, USA), and fluo - rescence intensities were measured using a multimode A ™ microplate reader (Spark 10 M, Tekan, Männedorf, Dye degradation eﬃciency (%) = 1 − × 100 Switzerland) at excitation (ex.) and emission (em.) wave- (1) lengths of 485 and 520 nm, respectively. where A is initial absorbance of the dye solution and A 0 t is dye absorbance during reaction at time t. Collection and in vitro maturation (IVM) of pig oocytes Ovaries were collected from prepubertal gilts at a local Boar sperm preparation and sperm incubation slaughterhouse. Cumulus–oocyte complexes (COCs) Liquid boar semen was purchased from a local artificial were aspirated from antral follicles (3–6 mm in diam- insemination (AI) center. The diluted semen was stored eter), washed three times in HEPES-buffered Tyrode in a storage unit at 17 °C for 5 days. Stock solutions lactate (TL-HEPES-PVA) medium supplemented with (1 mg/ml) of TiO NRs, Ag-TiO NRs, and Au-TiO NRs 0.01% (w/v) PVA, and then washed three times with 2 2 2 were prepared by suspension in phosphate-buffered solu - oocyte maturation medium (Abeydeera et al. 1998). A tion (PBS) and sonicated for 10 s at 60 Hz (Daihan Scien- total of 50 COCs were transferred to 500 µl of matura- tific, Korea) before use. For the incubation experiments, tion medium and layered with mineral oil in a 4-well Yi et al. Journal of Analytical Science and Technology (2023) 14:7 Page 4 of 14 multi-dish equilibrated at 38.5 °C in 5% CO in air. The DAPI and 10 μg/ml PNA for 40 min, and then observed oocyte maturation medium used was tissue culture under a fluorescence microscope (Nikon). medium (TCM) 199 supplemented with 0.1% PVA, 3.05 mM D-glucose, 0.91 mM sodium pyruvate, 0.57 mM Observations of sperm incubated with TiO NRs by TEM cysteine, 0.5 µg/ml luteinizing hormone, 0.5 µg/ml fol- Spermatozoa incubated with TiO NRs, Ag-TiO NRs, 2 2 licle-stimulating hormone, 10 ng/ml epidermal growth or Au-TiO NRs were fixed in modified Karnovsky’s fixa - factor, 75 µg/ml penicillin G, and 50 µg/ml streptomycin. tive (2% paraformaldehyde and 2% glutaraldehyde in Oocytes were cultured in TCM199 for 44 h at 38.5 °C, 5% 0.05 M sodium cacodylate buffer (pH 7.2) at 4 °C over - CO in air. night, washed three times with 0.05 M sodium cacodylate buffer at 4 °C for 10 min, and postfixed in 1% osmium In vitro fertilization (IVF) and culture (IVC) of pig oocytes tetroxide in 0.05 M sodium cacodylate buffer for 90 min. After IVM, cumulus cells were removed by treating them Fixed cells were washed twice with DI water at RT and with 0.1% hyaluronidase in TL-HEPES-PVA medium stained using 0.5% uranyl acetate at 4 °C overnight. Fur- (Abeydeera et al. 1998). Oocytes were then placed into ther, fixed cells were dehydrated using an increasing eth - four 100 μl drops of modified Tris-buffered medium anol series (30, 40, 50, 70, 80, 90, and 100%), embedded in (mTBM) in a 35-mm polystyrene culture dish and cov- EMbed 812 resin mixture containing DDSA, NMA, and ered with mineral oil. Spermatozoa were incubated in DMP-30, polymerized at 60 °C for 48 h, and ultra-thin BTS in the absence (W/O) or presence of TiO NRs (con- sections were stained with 2% uranyl acetate for 45 min trols), Au-TiO NRs, or Ag-TiO NRs (final conc.: 10 or followed by lead citrate for 3 min. TEM was conducted 2 2 20 μg/ml) for 2 h at 38 °C and washed twice in PBS con- using a Hitachi H-7650 unit at 80 kV (Tokyo, Japan). taining 0.1% PVA (PBS-PVA) at 800× g for 5 min. At the end of the washing procedure, sperm were resuspended Statistical analysis in mTBM, appropriately diluted, and sperm suspensions All experimental data were expressed as mean ± standard (1 µl) were added to medium containing oocytes to a final error of the mean (SEM), and analyzed using one-way 5 ® sperm concentration of 1 × 10 spermatozoa/ml. Oocytes ANOVA in GraphPad PRISM (GraphPad software, San were co-incubated with spermatozoa for 5 h at 38.5 °C Diego, CA, USA). The completely randomized design in a 5% CO atmosphere. After IVF, oocytes were trans- was applied, and Tukey’s multiple comparison test was ferred to 500 μl porcine zygote medium (PZM-3; Yosh- performed to compare values of individual treatments. ioka et al. 2002), supplemented with 0.4% bovine serum Results are considered statistically significant at *p < 0.05, albumin, and cultured for an additional 20, 48, or 144 h. **p < 0.01 and ***p < 0.001. The IVM, IVF, and IVC studies were repeated five times for each treatment regimen. Results Characterizations of noble metal‑loaded TiO NRs Fluorescence staining of oocytes and spermatozoa The XRD patterns of TiO NRs are shown in Fig. 1A. Oocytes/embryos were fixed with 2% formaldehyde for Major diffraction peaks at 2θ = 27.5, 36.1, and 54.4° cor- 40 min at room temperature (RT), washed twice with respond to (110), (101), and (211) crystal planes, which PBS, permeabilized with PBS-Triton X-100 for 30 min, is the most reported phase of rutile (JCPDS 89-4202) and stained with 2.5 mg/ml 4′,6-diamidino-2-phenylin- (Dhandole et al. 2016). However, small length NRs (or dole (DAPI; DNA staining; Molecular Probes, Eugene, broken residuals) were also observed which contributed OR, USA) for 40 min. The fertilization statuses of zygotes small portion in the overall synthesized product. The (unfertilized, fertilized-monospermic, or fertilized-pol- elemental analysis EDS is shown in Fig. 1E–H and rep- yspermic), cleaved embryo numbers, blastocyst forma- resents the elemental quantifications and noble metals (1 tion, cell number per blastocyst were determined under wt%) observed on the surface of T iO NRs. Particle size a fluorescence microscope (Nikon Eclipse Ci micro - distribution histogram for TiO NR and deposited noble scope; Nikon Instruments Inc., Seoul, Korea). To observe metals (Au and Ag) were illustrated in Fig. 1I, J. Ag and the attachment of TiO NRs to spermatozoa, TiO NRs Au particle size was determined by TEM analysis. 2 2 (1 mg/ml) were mixed with 1 mM alizarin red S (ARS) As shown in Fig. 2A (a, b), Ag and Au NPs were and stored at 4 °C until required (ur Th n et al. 2009). observed on the surfaces of TiO NRs and both had a Spermatozoa (1 × 10 /ml) were incubated in BTS in the diameter of 20–30 nm as determined by TEM. The oxi - presence of T iO NRs-ARS for 2 h at 38.5 °C. Spermato- dation states of elements and the chemical compositions zoa were then fixed with 2% formaldehyde for 40 min at of the as-synthesized materials were determined by XPS. RT, washed with PBS three times, stained with 2.5 μg/ml Figure 2B shows the high-resolution XPS spectra for Ti Yi et al. Journal of Analytical Science and Technology (2023) 14:7 Page 5 of 14 Fig. 1 Characteristics of novel metal‑loaded TiO NRs. A XRD image of rutile TiO NRs. FESEM images of B bare TiO NRs, C Ag‑loaded TiO NRs, and 2 2 2 2 D Au‑loaded TiO NRs. EDS spectrum and elemental quantification table (inset) of E TiO ‑NRs, F 2‑time washed TiO ‑NRs, G Au (1wt%)‑ TiO ‑NRs, and 2 2 2 2 H Ag (1wt%)‑ TiO ‑NRs. Particle size (length) distribution histogram for I TiO NR, and J deposited noble metals (Au and Ag) 2 2 Yi et al. Journal of Analytical Science and Technology (2023) 14:7 Page 6 of 14 Fig. 2 Nanoparticle attachment to TiO NRs as determined by electron microscopy. A TEM images of (a) silver (Ag) nanoparticle‑loaded TiO 2 2 NRs and (b) gold (Au) nanoparticle‑loaded TiO NRs. Dotted circles show Ag and Au nanoparticles on the surfaces of TiO NRs. (c) and (d) show 2 2 respective TEM maps. B High‑resolution XPS spectra of Ti 2p, O 1s, Ag 3d, and Au 4f oxidation peaks of (a) bare TiO NRs, (b) Ag‑loaded TiO NRs, and 2 2 (c) Au‑loaded TiO NRs 2 Yi et al. Journal of Analytical Science and Technology (2023) 14:7 Page 7 of 14 2p and O 1s of T iO NRs and the noble metal-loaded These results indicate that Ag-TiO NRs at 10 and 20 μg/ 2 2 TiO NRs. XPS peaks at around 458.5 and 464.0 eV cor- ml adversely affected the viability of spermatozoa, and respond to Ti 2p and Ti 2p spin–orbit pairs, respec- the higher decrement obtained for 2 h incubation sig- 3/2 1/2 tively, confirming that titanium doublet peaks were due nifies that sperm movement might be disturbed under to the Ti (IV) oxidation state (Li et al. 2005; Ohno et al. high doses of Ag-TiO NRs (p < 0.05 and p < 0.01; Fig. 4B). 2003). The peak at ~ 529 eV was ascribed to oxygen. The Regarding acrosomal integrity, there was a high percent- shifts of Ag and Au peaks to lower energies compared age of intact acrosome spermatozoa (PNA-/PI-) in the no with bulk material confirmed large quantities of both treatment sample, but damaged acrosomes significantly metals deposited on the surfaces of T iO NRs and that increased in the 20 μg/ml Ag-TiO NR group after incu- 2 2 strong metallic interactions had formed between noble bation of 30 min or 2 h than in the other groups (p < 0.05, metal NPs and the TiO NRs. The XPS spectra of Ag p < 0.01 and p < 0.001; Fig. 4C, D). To understand the 3d and Au 4f contained doublet peaks located at ~ 367.7 effects of catalysts on the motility and viability of sperma - and ~ 373.5 eV, which corresponded to the reported bind- tozoa, we measured intracellular ROS levels after treat- ing energies of Ag 3d and Ag 3d , respectively, and ing sperm for 2 h. Interestingly, ROS levels were found 5/2 3/2 peaks at ~ 83.0 and ~ 87.05 eV, which corresponded to to be significantly higher after treatment with Ag-TiO the binding energies of Au 4f and Au 4f , respectively NRs than for the other treatments (p < 0.05 and p < 0.001; 7/2 5/2 (Su et al. 2012; Zhang et al. 2014; Haruta 1997; Ma et al. Fig. 4E). 2014). Spermatozoa were incubated with ARS-coated TiO NRs (controls, Ag-loaded, and Au-loaded) for 4 h, Sperm motility in the presence of noble metal‑loaded TiO washed twice with PBS-PVA, fixed, and stained with NRs PNA (sperm acrosome, green) and DAPI (DNA, blue; We assessed sperm motilities using a CASA system; A–D); all NRs showed sufficient ARS (red fluorescence; motility parameters after treatments for 10 min and 2 h Fig. 5). Scattered or clumped coated T iO NRs (stained are provided in Fig. 3. The percentage of total motile red) were attached to sperm heads, midsections, or tails sperm after 10 min of incubation was higher for sperm (white arrows) after each treatment (Fig. 5A–D). In TEM treated with 10 μg/ml of Au-TiO NRs than for sperm analysis, Ag-TiO NRs and nanoparticles were observed 2 2 treated with 20 μg/ml of Ag-TiO NRs; however, differ - in acrosomal membrane (white arrows; Fig. 5E) (Lan and ences between treatment groups were not significant Yang 2012; Lafuente et al. 2016; Li et al. 2005, 2014; Ma (Fig. 3A, B). After incubation for 2 h, motility was signifi - et al. 2014). TiO NRs attached to spermatozoa were also cantly greater for non-treated controls than in the other observed during IVF, which presumably would prevent groups, and the motility of sperm incubated with 20 μg/ sperm penetrating oocytes (Fig. 5F). ml of Ag-TiO NRs was significantly less than in the other groups (*p < 0.05, **p < 0.01 and ***p < 0.001; Fig. 3C). The Fertilization and embryo development rates on sperm percentage of progressive motile spermatozoa was higher incubated with TiO NRs after treatment with 10 μg/ml Au-TiO NRs for 10 min, To determine fertilization rates, oocytes were insemi- but then decreased after treatment for 2 h. In particular, nated with spermatozoa incubated with controls and TiO we observed significant progressive loss of motility after NRs, Au-TiO NRs, or Ag-TiO NRs for 2 h (Fig. 6A). The 2 2 treatment with 20 μg/ml Ag-TiO NRs (**p < 0.01 and total fertilization rate including the rate of monospermic ***p < 0.001; Fig. 3D). Motility refers to the ability of sper- and polyspermic oocytes was lower when spermatozoa matozoa to move and swim independently in the female were incubated with 20 μg/ml Ag-TiO NRs (monosper- reproductive tract and is essential for successful ferti- mic: 33.9%, polyspermy: 0%) or 20 μg/ml Au-TiO NRs lization. These results suggest that Ag-TiO NRs affects (monospermic: 33.4%, polyspermy: 0%) than controls sperm movement either physically or chemically. (monospermic: 46.9–64.4%, polyspermy: 12.4–16.7%) and other treatments (monospermic: 41.5–54.8%, poly- Sperm viability, acrosomal integrity, and ROS levels spermy: 0–15.8%), but there were no significantly dif - in spermatozoa incubated with TiO NRs ferences among groups (Fig. 6A). As regards embryonic Figure 4 shows viability and intact acrosome results for development, we observed more cleaved oocytes from sperm incubated for 30 min or 2 h with T iO NRs, Ag- IVF performed using control sperm (83.1%) and a sig- TiO NRs, or Au-TiO NRs. Percentages of viable sperm nificantly lower cleavage rate when sperm were incubated 2 2 were higher after treatment with 10 μg/ml Au-TiO NRs with 20 μg/ml Ag-TiO NRs (54.1%) or 20 μg/ml Au-TiO 2 2 2 and for controls than after treatment with 20 μg/ml Au- NRs (55.6%) than in the other groups (60.6–75.1%, p < 0.05; TiO NRs 10 or 20 μg/ml Ag-TiO NRs (75.7–76.3% vs. Fig. 6B). Furthermore, the blastocyst formation rate was 2 2 63.0–73.6%; p < 0.05, p < 0.01 and p < 0.001; Fig. 4A). higher in the control group (27.1%) than in the other Yi et al. Journal of Analytical Science and Technology (2023) 14:7 Page 8 of 14 Fig. 3 Comparison of total motile sperm and progressive motile sperm after incubation with or without ( W/O) TiO NRs. Boar spermatozoa were incubated in the presence of TiO 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 groups (10.2–4.1%; Fig. 6C), and when oocytes were ferti- the degradation of organic orange II dye in the presence lized with spermatozoa incubated with 20 μg/ml Ag-loaded of Ag-TiO NR catalyst. Orange (II) dye contains an azo TiO NRs, the lowest blastocyst rates were observed (2.2%, linkage, which forms N=N bonds with its chromophores p < 0.05 and p < 0.001; Fig. 6C). However, mean cell number benzene and naphthalene, and this linkage is sensitive per blastocyst in the groups were not significantly different to active radical species such as OH·, HOO·, and O· (30.1–45.0 cells/blastocyst). (Dhandole et al. 2017). We observed low dye concentra- tions in the presence of 0.05–2 mM NADPH and 0.5 mg/ Intracellular ROS generation by Ag‑TiO NRs ml Ag-TiO NRs (Fig. 7A), but single treatments with 2 2 in spermatozoa NADPH or Ag-loaded T iO NRs had no effect. This result To investigate intracellular ROS generation in sperm indicates that the synergistic effect of NADPH and Ag- exposed to Ag-TiO NR, we first examined the role of TiO NR treatment for 4 h increased the dye degradation 2 2 NADPH during the process of electron donation. Spe- rate as increasing concentration of NADPH (Fig. 7A). cifically, we tested the effects of NADPH activity over Based on this result, we fixed the concentration of Yi et al. Journal of Analytical Science and Technology (2023) 14:7 Page 9 of 14 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 TiO 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 TiO NRs, C Ag‑loaded TiO NRs, and D Au‑loaded TiO NRs for 4 h. E TEM image of TiO NRs attached to sperm acrosome 2 2 2 2 and nuclear membrane (white arrows). F TiO NRs attached to spermatozoa inhibited sperm penetration during IVF 2 Yi et al. Journal of Analytical Science and Technology (2023) 14:7 Page 10 of 14 Fig. 6 Fertilization competence of sperm incubated with/without TiO NRs. A Oocytes were fertilized with sperm incubated with control ( W/O), Ag‑loaded, and Au‑loaded TiO 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 NADPH at 0.2 mM and conducted the same experiment Discussion but replaced the organic dye with fresh sperm (Fig. 7B). Remarkable advances in nanotechnology have made We observed higher ROS levels when NADPH (0.2 mM) many novel biomedical applications possible, espe- and Ag-loaded TiO NRs were treated in combination; cially in the reproductive biology field. However, due to ROS production was low when only NADPH was admin- the environmental effects of the widespread use of NPs istered (Fig. 7B). These results indicate the importance and their adverse effects on animal germ cells, their use of NADPH as an electron donor (Aitken et al. 1997). In should be subjected to strict review (Falchi et al. 2018). the presence of low NADPH levels, Ag-loaded T iO NRs In particular, NPs have been recently reported to be toxic show enhanced generating of ROS, which suggests these to male reproductive organs and germ cells. Small NPs NRs acted as charge carriers from NADPH donor ligands easily penetrate cell membranes, such as the blood–testis (Fig. 8). barrier, accumulate or deposit in testis, and disrupt sperm formation and development (Lan and Yang 2012). In rats Yi et al. Journal of Analytical Science and Technology (2023) 14:7 Page 11 of 14 Fig. 7 ROS generation in spermatozoa incubated with Ag‑loaded TiO NRs using NADPH. A Dye degradation of Ag‑loaded TiO NRs in the presence 2 2 of NADPH. B ROS generation (in sperm) incubated with different catalysts Ag nanoparticles, TiO NRs, and Ag‑ TiO NR in the presence of commercial 2 2 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 TiO NR catalyst 2 Yi et al. Journal of Analytical Science and Technology (2023) 14:7 Page 12 of 14 fed Ag NPs, sex hormone levels decreased and abnormal reactions have been disrupted. The affinity of the Ag sperm morphology and motility increased (Neill et al. metallic state is higher than the donor ligand, and reduc- 2009; Baki et al. 2014; Lafuente et al. 2016), and admin- tion potential is a parameter of thermodynamic reactions istering Au NPs to mice caused abnormal sperm chroma- (Vernet et al. 2001; Prasad et al. 2017). The reduced spe - tin remodeling and DNA damage (Nazar et al. 2016). In cies, Ag , transfer electrons to the conduction band of similar experiments, functional defects and DNA damage TiO because the band edge of the TiO is lower than that 2 2 in spermatozoa were observed in male mice administered of the Ag-loaded NPs (as shown in Fig. 7; Kochuveedu TiO NPs (Smith et al. 2015), and direct exposure to NPs et al. 2013). After an electron is released, Ag re-oxidizes, interfered with sperm function, for example, bull sperm and the cycle continues until the donor ligand actively exposed to Au NPs showed impaired motility and fertil- participates in the reaction. Meanwhile, conduction band ity (Taylor et al. 2014), and human sperm treated with electrons reduce oxygen molecules to form OH·, O , 44 ppm of Au NPs showed 25% lower motility than non- and OOH· radicals (Dhandole et al. 2017), which cause treated controls (Wiwanitkit et al. 2009). In the present cell damage and DNA fragmentation. In a recent study, it study, direct exposure of Ag-loaded T iO NRs to boar was reported that morin and rutin have protective effects spermatozoa decreased motility, viability, and acrosomal on rat testis from oxidative damage caused by ROS asso- integrity, which resulted in lower fertilization and embry- ciated with T iO NP intake (Hussein et al. 2019). There - onic development rates. fore, a further study on antioxidants to mitigate the Spermatozoa are highly sensitive cells dedicated to fer- toxicity and oxidative damage associated with ROS pro- tilizing oocytes, and many factors can influence sperm duction derived from Ag- or TiO2 NRs is needed in the attachment and fertilization. The most problematic fac - future. tor is the intracellular production of ROS and related oxi- dative stress, which adversely affects sperm survival and Conclusions fertility (Saadeldin et al. 2020). Vernet et al. (2001) sug- In the present study, Ag or Au NPs were incorporated on gested that intracellular ROS generation might be a col- the surfaces of T iO NRs by photo-deposition, and their lective effect of the mitochondrial respiratory chain and effects on boar spermatozoa were examined. In addi - NADPH oxidase system in sperm plasma membranes. tion, porcine oocytes matured in vitro were inseminated The mitochondrial electron transport chain reaction pro - with sperm that had been incubated in the presence of duces ROS under physiological conditions, and this is the noble metal-loaded NRs, and fertilization rates and correlated with physical activity (Riaz et al. 2017; Vernet embryo developments were investigated to verify germ et al. 2004). However, recent studies have confirmed that cell toxicity in preimplantation embryos. Our experi- the cytotoxic effect of Ag NPs can also induce intracel - mental results indicated that Ag-TiO NRs at 20 μg/ml lular ROS production, which is responsible for most of generated ROS that decreased the fertilization rate and the common abnormalities in spermatozoa, such as dis- reduced sperm viability and acrosome integrity. Further- rupted chromatin, bent tails, curved midsections, DNA more, our investigation of the charge transfer mechanism fragmentation, mitochondrial damage, respiratory chain and ROS generation showed that a catalytic redox reac- disruption, oxidative stress, and chromosomal aber- tion took place in sperm nuclei and that mitochondrial rations (Haase et al. 2012; Beer et al. 2012; Wang et al. NADPH functions as an electron donor and activates Ag- 2017). loaded TiO NRs. Based on a literature review, we report an electron transfer mechanism that underlies spermatozoa inactiva- Acknowledgements Y‑ JY was supported by National Research Foundation of Korea (NRF) Grants tion and present for the first time the effect metal oxide funded by the Korea government (MSIT ) (NRF‑2013R1A6A3A04063769 and nanoparticles have on intracellular ROS stress in sperma- NRF‑2020R1A2C1014007), and JSJ was supported by research funds of Jeon‑ tozoa caused by mitochondrial transmembrane electron buk National University in 2022. transport. We present the suggested general electron Author contributions transfer mechanism in a schematic diagram in Fig. 8. The Y‑ JY, and LKD have contributed equally to this work. Y‑ JY, LKD, and JSJ contrib‑ physical contact of sperm cell with Ag-loaded TiO NR 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 will result in Schottky barrier whose Fermi-level align- in writing—original draft. Y‑ JY, LKD, and JSJ contributed to writing—review ment (based on band theory) results in the facile electron and editing. All authors read and approved the final manuscript. transport from spermatozoa to Ag NPs over the surface Funding of TiO NRs. The electron donor ligand of Ag NP might This work was supported by National Research Foundation of Korea (NRF) accept the transmembrane electrons of sperm enzymatic Grants funded by the Korea government (MSIT ) (NRF‑2013R1A6A3A04063769 NADPH after mitochondrial electron transport chain and NRF‑2020R1A2C1014007). Yi et al. Journal of Analytical Science and Technology (2023) 14:7 Page 13 of 14 Availability of data and materials Hussein MM, Gad E, Ahmed MM, Arisha AH, Mahdy HF, Swelum AA, Tukur Upon reasonable request, the datasets of this study can be available from the HA, Saadeldin IM. 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Journal of Analytical Science and Technology – Springer Journals
Published: Jan 26, 2023
Keywords: TiO2 nanorods; Noble metals; In vitro fertilization; NADPH; Embryo development; Au; Ag; Spermatozoa
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