Influence of nanomaterials on mammalian spermatozoa and testes

Metals in general have been demonstrated to harm male fertility even at very low concentrations (PIZENT et al. 2012). Compared to the female side more studies with nanoparticles are related to sperm, probably due to the much easier access to ejaculates than to oocytes and the well-defined fertility parameters of sperm, which are regularly used for semen evaluation. For example, MORETTI et al. (2013) incubated human ejaculated spermatozoa with AuNP or AgNP and observed a significant decrease in motility and viability after 60 min compared to the control population at the same time. The according examination using transmission electron microscopy revealed the attachment of AuNP to the sperm surface, whereas no membrane association of the AgNP could be detected. The authors hypothesize that the impairment resulting from the co-incubation with AgNP might be due to the ion release of the nanoparticles. The findings concerning the AuNP were confirmed in a similar study performed by TAYLOR et al. (2014a), who incubated bovine sperm with AuNP, which were either ligand-free or conjugated with oligonucleotides. After a 2 h exposure no difference in membrane integrity or sperm cell morphology was observed, but a significant decrease in motility occurred. They found no intact sperm being penetrated by NP, but the ligand-free NP showed association to the outer sperm surface. Apparently, a motility loss can already occur after a much shorter exposure time, as shown by WIWANITKIT et al. (2009), who incubated human spermatozoa with AuNP for only 15 min. This underlines the hypothesis that the decrease in motility is a result of the physical overload of the spermatozoa through the NP, as supposed by all above mentioned authors. Boar sperm motility is also reduced by nanomaterials like quantum dots (QD) within 30 min of incubation (FEUGANG et al. 2012) or europium-oxide nanoparticles (Eu2O3NP) incubated with bovine spermatozoa for 24 h (MAKHLUF et al. 2008).

Chromatin decondensation is necessary for successful fertilization. The ability of chromatin to decondense can be tested in vitro and very small AuNP have been shown to hinder this process in mouse epididymal sperm incubated with 2.5 nm AuNP (ZAKHIDOV et al. 2010) and also in frozen-thawed bovine sperm incubated with 3 nm AuNP (ZAKHIDOV et al.

2013). Even DNA damage is a possible result of the exposure of sperm to nanoparticles, as shown by GOPALAN et al. (2009), who incubated human spermatozoa with titanium-dioxide

(TiO2NP) or zinc-oxide nanoparticles (ZnONP). A concentration dependency of this effect was clearly detected, but already very low concentrations seem to have this effect as described by PREAUBERT et al. (2015) on mice sperm incubated with cerium dioxide nanoparticles.

The direct impairment of sperm, as determined by the above-mentioned in vitro assays, already raises high concerns, regarding nanoparticle exposure and male reproductive function, but these direct effects to sperm cell viability could so far only be shown in vitro.

Damage to the testis can be caused by NP uptake and distribution through the body. For example, in mice the intra-tracheal exposure to carbon nanoparticles, which are present in ambient air and are generated during combustion processes, led to migration of these particles into the testes, caused vacuolation of the seminiferous tubules, and resulted in a decrease of daily sperm production (YOSHIDA et al. 2009). Amorphous nanosilica particles (70 nm) also entered the testes of mice after intravenous injection and were found in Sertoli cells, cytoplasm, and nucleus of spermatocytes, while larger silica particles (300 nm) administered the same way could not be detected in the testes (MORISHITA et al. 2012). Nickel nanoparticles (NiNP) daily administered to male rats for ten weeks by gavage led to increased epithelial cell shedding, disordered cell arrangement, and increased cell apoptosis in the testes (KONG et al. 2014). These findings demonstrate that especially chronic contact, for example with the inevitable inhalation of ambient air or continuing exposure in the work environment, can seriously influence the male reproductive performance in mammals. This has also been observed by ANTONINI (2003), who noted that there is a higher incidence of infertility in male welders compared to other males of the same age and could trace this back to nanoparticle exposure.

Table 2: Provided nanoparticle (NP) characteristics of the above described studies on male reproductive function and nanoparticle effects. Highlighted are studies observing adverse effects. Considerations are also given in regard to comparability (QD: quantum dots, PVP: polyvinylpyrrolidone, PVA: polyvinylalcohol). Type of study/ SpeciesMaterialCoating SizeDoseAdministration/ ExposureObtained Parameters Adverse effect? ReferenceConsiderations In vitro/ human AuNP ? 50 nm 30, 60, 125, 250, 500µMEjaculated sperm, 60 min Motility and viability Yes (125µM and more)(MORETTI et al. 2013)

Coating and dose per cell not provided AgNP65 nmYes (125µM and more) In vitro/ bovineAuNPnot coated 10.8 nm0.1, 1, 10µg per 100x106 sperm (in 1 ml)

Ejaculated sperm, 120 min (37 °C) Motility, membrane integrity, morphology

Yes (motility for 10µg/ml)(TAYLOR et al. 2014a) Comparable Oligo- nucleotides 7.3 nm In vitro/ humanAuNP? 9 nm? Ejaculated sperm, 15 min MotilityYes (WIWANITKIT et al. 2009)

One sperm donor; dose and coating not provided In vitro/ porcine CdSe/ ZnsQDs

Renilla luciferase/ nona- arginin peptide

5-7 nm

1 nM per 0.1, 0.5, 1, 2x108 sperm (in 1 ml) Ejaculated sperm, 30 min

MotilityYes (0.1x108 sperm) (FEUGANG et al. 2012)Comparable 1, 5 nM per 1x108 sperm (in 1 ml) Viability No In vitro/ bovineEu2O3NPnot coated 30 nm 2.5 mg/mlEjaculated sperm, 24 hMotilityYes (MAKHLUF et al. 2008)Comparable PVP9 nmNo PVA15 nmNo In vitro/ murineAuNPnot coated 2.5 nm

0.5x1015 or 1x1015 particles/ml (diluted) Epididymal sperm, 20 min Chromatin decondensation ability Yes (ZAKHIDOV et al. 2010)Dose per cell not provided

Table 2 continued Type of study/ SpeciesMaterialCoating SizeDoseAdministration / ExposureObtained Parameters Adverse effect? ReferenceConsi In vitro/ bovineAuNPnot coated 3 nm1x1015 particles/ml (diluted) Ejaculated sperm, 20 min or 40 min

Chromatin decondensation ability Yes (ZAKHIDOV et al. 2013)Dose not In vitro/ human

TiO2NP ? 40-70 nm3.7-59.7 µg/ml Ejaculated spermDNA damage (comet assay) Yes (GOPALAN et al. 2009)

Expo dos and co prZnONP11.5-93.2 µg/ml In vitro/ murineCeO2NPnot coated 7 nm0.01 mg/l to 1.15 sperm/ml (in 200µl)

Epididymal sperm for 60 min

DNA damage (comet assay) Yes (PREAUBERT et al. 2015)Com In vivo/ murineCarbon NP? 14 nm200µg/mouse 2x during gestationMale mice in utero Testis tissue integrity and daily sperm production

Yes (YOSHIDA et al. 2009)Co pr In vivo/ murineSilicaNPnot coated 70 nm0.8 mg on two consecutive days

Distribution through blood stream after injection

Penetration of sertolli cells and spermatocytesYes (MORISHITA et al. 2012)Com In vivo/ murineNiNP? 90 nm 5, 15, 45 mg/kg/day (10 weeks) Gavage

Testis weight Yes (45 mg/kg) (KONG et al. 2014)SiCo pr

Epididymis weight

Yes (15, 45 mg/kg) Motility parameters

Yes (15, 45 mg/kg) Testis tissue damageYes (45 mg/kg)

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