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Effects of Prepubertal Exposure to Silver Nanoparticleson Reproductive Parameters in Adult Male Wistar RatsHanan Khaled Sleiman a , Renata Marino Romano b , Claudio Alvarenga de Oliveira c & MarcoAurelio Romano aa Department of Pharmacy , State University of Centro-Oeste , Parana , Brazilb Section of Experimental Endocrinology, Department of Pharmacology , UniversidadeFederal de São Paulo , São Paulo , Brazilc Department of Animal Reproduction , Veterinary Medicine School, University of São Paulo ,São Paulo , BrazilPublished online: 29 Oct 2013.

To cite this article: Hanan Khaled Sleiman , Renata Marino Romano , Claudio Alvarenga de Oliveira & Marco Aurelio Romano(2013) Effects of Prepubertal Exposure to Silver Nanoparticles on Reproductive Parameters in Adult Male Wistar Rats, Journalof Toxicology and Environmental Health, Part A: Current Issues, 76:17, 1023-1032, DOI: 10.1080/15287394.2013.831723

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Journal of Toxicology and Environmental Health, Part A, 76:1023–1032, 2013Copyright © Taylor & Francis Group, LLCISSN: 1528-7394 print / 1087-2620 onlineDOI: 10.1080/15287394.2013.831723

EFFECTS OF PREPUBERTAL EXPOSURE TO SILVER NANOPARTICLES ONREPRODUCTIVE PARAMETERS IN ADULT MALE WISTAR RATS

Hanan Khaled Sleiman1, Renata Marino Romano2, Claudio Alvarenga de Oliveira3,Marco Aurelio Romano1

1Department of Pharmacy, State University of Centro-Oeste, Parana, Brazil2Section of Experimental Endocrinology, Department of Pharmacology, Universidade Federalde São Paulo, São Paulo, Brazil3Department of Animal Reproduction, Veterinary Medicine School, University of São Paulo,São Paulo, Brazil

The incidence of male reproductive pathologies, such as hypospadias, cryptorchidism, tes-ticular cancer, and low sperm production in adulthood, is increasing and may be related toexposure to environmental contaminants. The silver nanoparticles (AgNP) are a new classof chemical compounds commonly used in both medical and nonmedical settings, and theyaffect development of spermatogonial stem cells in vitro. The aim of this study was to exam-ine the adverse productive toxic effects of AgNPs in male Wistar rats exposed during theprepubertal period and sacrificed at postnatal day (PND) 53 and PND90. Growth was assessedby daily weighing. The progress of puberty in the rats was measured by preputial separa-tion, while spermatogenesis was assayed by (1) measuring the sperm count in testes andepididymis and (2) examining the morphology and morphometry of seminiferous epitheliumusing stereological analysis. In addition, testosterone and estradiol levels were assayed byradioimmunoassay. The weight of the animals at PND90 did not change markedly, but growthwas less in the group treated with AgNP at 50 µg/kg from PND34 to PND53. AgNP expo-sure produced a delay in puberty in both treated groups. Decreased sperm reserves in theepididymis and diminished sperm transit time were observed at PND53, while a reduction insperm production occurred at PND90. The morphology of the seminiferous epithelium wasmarkedly altered. Data demonstrated that prepubertal exposure to AgNP altered reproductivedevelopment in prepubertal male Wistar rats, as evidenced by impairment in spermatogenesisand a lower sperm count in adulthood.

Silver nanoparticles (AgNP) are a class ofparticles that vary in size (1 to 100 nm) andshape, which affects their physical character-istics. Consequently, these variations manifestas differences in biological activity and poten-tial toxicity of the particles (Schluesener andSchluesener, 2013). The AgNP are commonlyused in medical practice because of their antivi-ral, antifungal, and antibacterial properties fortreatment of wounds and burns, as well asantibacterial surface coating. Further AgNP are

Received 8 June 2013; accepted 28 July 2013.H. K. Sleiman was the recipient of a scholarship from CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nivel Superior).Address correspondence to Marco Aurelio Romano, R. Simeao Camargo Varela de Sa, 03, Guarapuava, PR, CEP 85040-080, Brazil.

E-mail: maromano17@gmail.com

utilized in nonmedical purposes as a com-ponent of consumer products in toothpaste,disinfecting sprays, and food containers (Duránet al., 2010; Dhawan and Sharma, 2010;García-Contreras et al., 2011; Horie et al.,2012; Savithramma, 2011). Thus, the extensiveuse of AgNP increases the risk of environ-mental contamination (Panacek et al., 2011),and acute and chronic studies are necessaryto understand the effects of this novel class ofchemical compounds.

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It needs to be emphasized that it is necessaryto characterize the physical, chemical, and bio-logical properties of nanoparticles, in addition totheir toxicological effects, for a comprehensiverisk assessment (Horie et al., 2012; Snyder-Talkington et al., 2012). Howeverr, toxicologicalassessment of nanomaterials has produced con-tradictory results (Garcia-Alonso et al., 2011).Recent studies demonstrated the adverse effectsof AgNP including cytotoxicity (Asare et al.,2012), genotoxicity (AshaRani et al., 2009), inhi-bition of mitochondrial activity (Braydich-Stolleet al., 2005; Hadrup et al., 2012; Carlson et al.,2008),elevationofd the levelsofmetallothioneinand lipid peroxidation (Gagne et al., 2013), andproduction of reactive oxygen species (ROS),resulting in oxidative stress in the central ner-vous system (Win-Shwe and Fujimaki, 2011).In contrast, inhalation of AgNP (as found inspray products) by rats did not markedly affectseveral pulmonary and cardiovascular functionparameters (Roberts et al., 2013).

Reproductive physiology involves complexbiological processes that are sensitive to chemi-cal contaminants. The incidence of male repro-ductive pathologies classified as testicular dys-genesis syndrome, such as hypospadias, cryp-torchidism, testicular cancer, and low spermproduction in adulthood, is increasing. This risemay be due in part to exposure to environmen-tal contaminants (Lucas et al., 2009). In thiscontext, AgNP are known to affect the devel-opment of spermatogonial stem cells in vitro,and consequently this affects the spermatogen-esis process (Lucas et al., 2009; Braydich-Stolleet al., 2010; Asare et al., 2012) Using an invivo model may provide a better understand-ing of the effects of exposure to AgNP on malereproductive functions. The aim of this studywas thus to investigate the adverse reproductiveeffects of AgNPs on Wistar male rats exposedduring the prepubertal period.

MATERIAL AND METHODS

ChemicalsTreatment was performed with AgNP

suspension of 60-nm diameter particles

(reference number 730815, Sigma-Aldrich Co,Germany).

Experimental DesignThirty newly weaned male Wistar rats orig-

inating from female rats that were followedfrom d 17 of pregnancy, in order to deter-mine the exact days of birth, were used inthe experiment. On postnatal day 4 (PND4),litters were culled to 8 pups per female andkept at this proportion until weaning (PND21).Animals were maintained on rat diet ad libitum,under a 12-h dark/light cycle in a controlled-temperature room (23 ± 1◦C). Rats weresubmitted to experimental treatments fromPND23 until PND53 and sacrificed by cervi-cal decapitation at PND53 or PND90. AgNPwas diluted in a watery suspension and admin-istered once a day, orally by gavage, in a volumeof 0.25 ml/100 g body weight between 7 and8 a.m. Animals were randomly assigned toone of three groups receiving the followingdoses of AgNP: 0 (control), 15, or 50 µg/kgbody weight (BW). The doses of AgNP werebased on toxicological values obtained fromtherapeutic products using ionic Ag. The ini-tial dose of 5 µg/kg BW was 100-fold lowerthan the therapeutic dose (Samberg et al.,2010) and without effect. Thus, the initialdose was increased threefold to 15 µg/kgBW and 10-fold to 50 µg/kg BW, respec-tively. All procedures were in accordance withBrazilian College of Animal Experimentationand approved by the Universidade Estadual doCentro-Oeste–Ethical Committee for AnimalResearch (protocol 021/2011). The experimen-tal design was based on the protocol of Romanoet al. (2010).

Preputial Separation (PPS) and BodyGrowthIn order to assess the age at puberty,

the balanopreputial separation, which consistsof the separation of the preputial membraneand externalization of the glans of the penis,was determined (Korenbrot et al., 1977). Thismethod was performed by tissue manipulation

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at PND33 and continued once a day dur-ing the period of balanopreputial separation.During this period, animals were also weighedto determine growth.

Sperm EvaluationThe sperm count was determined as pre-

viously described (Romano et al., 2012; Robbet al., 1978). The testes and epididymis (caput,corpus, and cauda) were weighed. The tunicaalbuginea was removed from the testes, andremaining parenchyma was homogenized in5 ml saline–Triton 0.5% followed by sonicationfor 30 s. The samples were then diluted 10-foldin saline, and mature spermatids resistant tohomogenization were counted using a hemo-cytometer. The four chambers were averaged,and numbers of spermatid per testis and pergram testis were calculated. These values weresubsequently divided by 6.1 d to calculate thedaily sperm production (DSP). The segments ofthe epididymis (caput, corpus, and cauda) wereminced, homogenized, diluted, and counted asdescribed for testes. The number of spermato-zoa in each homogenate was determined, asalready described, and total numbers of sper-matozoa for different parts of the epididymiswere measured. The mean time for sperm tran-sit through the epididymis also was determinedby dividing the number of spermatozoa in eachportion of the epididymis by the DSP of theassociated testis.

Histology and Morphometry ofSeminiferous EpitheliumTestes were fixed in Bouin’s solution for 8 h,

treated with alcohol and embedded in paraffin,and pieces of 5 µm size were prepared andlaminae were stained with hematoxylin andeosin. The laminae were observed initially witha 40× magnification for general organ archi-tecture observation. Next, a magnification of100× was used for a more detailed analysisof the seminiferous tubule architecture, as pre-viously described (Romano et al., 2010). Thisincluded analyzing the linear morphometry ofthe seminiferous tubules by determining the

tubular diameter (measured from the basal lam-ina to the basal lamina in the opposite direc-tion), seminiferous epithelium length (from thebasal lamina to the neck of the elongated sper-matids), and luminal diameter. Ten fields persection per animal were selected within the his-tological sections in the transverse direction ofthe tubules. For each tubule, the means werecalculated for the measurements indicated, andthe mean of each field was also calculated. Themeasurement for each animal was obtained bymeasuring all analyzed fields.

Reproductive Organ WeightsThe testes, epididymides, and seminal vesi-

cles were weighed at PND53 and PND90. Theepididymis was previously divided into threesegments: caput, corpus, and cauda. The sem-inal vesicle was weighed with fluid (undrained)and after fluid removal (drained).

Hormone MeasurementsSerum hormone concentrations were mea-

sured by radioimmunoassay using commer-cial kits for total testosterone and estradiol(Testosterone Coat-A-Count and Estradiol Coat-A-Count, Siemens Health Care Diagnostics,Los Angeles, CA). The intra-assay coefficientwas <5.8% for testosterone and <2.0% forestradiol.

Statistical AnalysisThe variables were first submitted to

Kolmogorov–Smirnov tests for normality andBartlett’s test for homoscedasticity. Analysis ofbody growth was performed using multipleanalysis of variance for repeated measures(MANOVA) by a general linear model (GLM).The weights were compared between differ-ent groups and ages, considering the expectedchanges with age. The day of PPS was com-pared among the groups using nonparametricanalyses with the Kruskal–Wallis method fol-lowed by the post hoc Dun test. All other

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parameters were analyzed by analysis of vari-ance (ANOVA) followed by Tukey’s HSD (hon-est significance difference) test and performedwithin the same age group (PND53 or PND90).All analyses were performed with Statistica 7.0(Statsoft, Inc., Tulsa, OK). Statistical differenceswere considered significant when p < .05. Thevalues were expressed as means and standarderror of the mean (±SEM) for parametric andinterquartile ranges of nonparametric analysis.

RESULTS

Body Growth AssessmentThe initial weight at PND23 (58.2 ± 1.6 g,

59.7 ± 2.1 g, and 60 ± 2.1 g for rats treatedwith 0, 15, and 50 µg/kg BW AgNP, respec-tively) and the final weight at PND90 (380.1± 5.9 g, 358.9 ± 8.5 g, and 377.5 ± 14.3 gfor rats treated with 0, 15, and 50 µg/kg BWAgNP, respectively) were not markedly differ-ent. However, the growth measured by dailyweighing was significantly different betweenPND34 and PND53 in the group receivingAgNP 50 µg/kg. After this period, the weightwas similar between all groups (Figure 1).

The Effects of AgNP on Age and BodyWeight at PubertyThe progression of puberty was monitored

by daily inspection of the preputial separation.The beginning of puberty was delayed in bothAgNP-treated groups (Figure 2). The weight atpuberty was not affected.

Prepubertal Exposure to SilverNanoparticles Promotes Changes inSperm ProductionThe effects of AgNP were determined on

total sperm production, daily sperm produc-tion, sperm reserves, and sperm transit atPND53 and PND90. AgNP exposure duringthe prepubertal period produced a numericalreduction in total and daily sperm productionin the 50-µg/kg BW AgNP-treated group atPND53. At PND90, both AgNP-treated groups

had significantly lowered total and daily spermproduction (Table 1). AgNP exposure during theprepubertal period also decreased the spermreserves in the caput, corpus, and cauda ofthe epididymis in both treatment groups atPND53 and PND90 (Table 1). The sperm tran-sit time through the segments of the epididymiswas significantly reduced by AgNP treatment atPND53 in both groups (Table 1).

Influence of Prepubertal AgNP Exposureon Morphology and MorphometryMorphometric analysis of the seminiferous

tubules did not reveal marked alterations inepithelial height or in luminal and tubular diam-eters. At PND53, the group treated with AgNPat 15 µg/kg showed quantitative reduction inthese parameters, but at PND90 these valueswere similar to control (Table 2).

The morphologic evaluation ofseminiferous epithelium revealed alterations.At PND53 and PND90, there were discon-tinuity and disorganization of seminiferousepithelium, cellular debris in the lumen,and sloughing of the germinal cells from theepithelium into the tubular lumen (Figure 3).

Prepubertal Exposure to AgNP Decreasesthe Weight of the EpididymisThe effect of prepubertal exposure to AgNP

during the prepubertal period was assessedbased on weight of testes, epididymis (caput,corpus, and cauda), seminal vesicles (drainedand undrained), and ventral prostate. Theweight of the cauda of the epididymis atPND53 was the only parameter affected, as evi-denced by a decrease in both groups receivingAgNP (Table 3).

Testosterone and Estradiol SerumConcentrationsThe measurement of testosterone and

estradiol serum concentrations did not revealany significant alterations at PND53 orPND90 exposed to AgNP (data not shown).

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FIGURE 1. Body growth in male rats exposed to AgNP during the prepubertal period. Data presented are mean ± SEM for control, 15 and50 µg/kg BW AgNP-treated groups. The arrows indicate the initial and final ages at which the weight of the 50-µg/kg BW AgNP-treatedgroup was significant compared to control (MANOVA, p < .05).

FIGURE 2. Age at puberty (PPS) in male rats exposed to AgNP during the prepubertal period. Data correspond to the median, theinterquartile range, and the non-outlier range for the control and the 15- and 50-µg/kg BW AgNP-treated groups. Asterisk indicatessignificant difference from control, p < .05 (Kruskal–Wallis followed by Dunn’s test).

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TABLE 1. Sperm Parameters at PND53 and PND90 in Male Rats Exposed to Silver Nanoparticles (AgNP) During Prepubertal Period

PND53, AgNP (µg/kg) PND90, AgNP (µg/kg)

Parameter Control 15 50 Control 15 50

Total sperm production (× 106/g testis) 67.5 ± 11.5 62.7.1 ± 4.4 48.2 ± 10 109.3 ± 14a 53.1 ± 4.4b 49.1 ± 5.1b

Total sperm production (× 106/ testis) 89.4 ± 15.2 79.2 ± 5.6 65.4 ± 14.6 175.2 ± 21.1a 90 ± 9.6b 78 ± 7.3b

Daily sperm production (× 106/g testis) 11.1 ± 1.9 10.3 ± 0.7 7.9 ± 1.6 17.9 ± 2.3a 8.7 ± 0.7b 8.1 ± 0.8b

Daily sperm production (× 106/ testis) 14.6 ± 2.5a 13.0 ± 0.9 10.7 ± 2.4 28.7 ± 3.5a 14.7 ± 1.6b 12.8 ± 1.2b

Sperm reserves∗Caput + corpus of epididymis (× 106) 29.2 ± 32a 13.2 ± 4.1b 4.2 ± 1.2c 171.9 ± 32a 68 ± 8.5b 82.8 ± 19.2b

Cauda of epididymis (× 106) 27.6 ± 6.2a 9 ± 4.1b 3 ± 0.1c 479.4 ± 66a 261 ± 33.2b 254.4 ± 40.8b

Sperm transit time throughCaput + corpus of epididymis (d) 1.5 ± 0.4a 0.5 ± 0.1b 0.3 ± 0.1b 2.6 ± 0.3 2.8 ± 0.5 3.2 ± 0.6Cauda of epididymis (d) 1± 0.2a 0.4 ± 0.2a 0.3 ± 0.2b 8.5 ± 0.7 9.2 ± 1.3 11.1 ± 0.7

Note. Data are mean ± SEM. Asterisk, two epididymides. Superscript a, b, and c differ in rows within the same age: p < .05. ANOVAand post hoc Tukey HSD; AgNP, silver nanoparticles.

TABLE 2. Morphometry of the Seminiferous Tubules at PND53 and PND90 in Male Rats Exposed to Silver Nanoparticles (AgNP) inPrepubertal Period

Epithelial height (µm) Luminal diameter (µm) Tubular diameter (µm)

PND53Control 97.8 ± 1.4 267.8 ± 14 463.4 ± 16.8AgNP 15 µg/kg 93.6 ± 1.2 257.8 ± 09.9 445.1 ± 11.3AgNP 50 µg/kg 98.3 ± 1.2 280.0 ± 04.4 476.6 ± 04.2

PND90Control 97.1 ± 1.1 305.4 ± 09.9 488.9 ± 11.7AgNP 15 µg/kg 97.6 ± 2.5 280.5 ± 11.4 475.7 ± 15AgNP 50 µg/kg 102.7 ± 1.3 289.3 ± 10.3 494.8 ± 10

Note. Data are mean ± SEM; AgNP, silver nanoparticles.

FIGURE 3. Morphology of the seminiferous epithelium of male rats exposed to AgNP during the prepubertal period: (A) control group atPND53, (B) 15 µg/kg BW AgNP-treated group at PND53, (C) 50 µg/kg BW AgNP-treated group at PND53, (D) control group at PND90,(E) 15 µg/kg BW AgNP-treated group at PND90, (F) 50 µg/kg BW AgNP-treated group at PND90. In (A), the lower case letters represent(a) spermatogonia, (b) spermatocyte, (c) elongated spermatids, (d) tubular lumen, and (e) interstitial space. The numbers in the panelsrepresent the following abnormalities: (1) sloughing of germinal cells, (2) germinal cells and cellular debris in the lumen, (3) discontinuityof the seminiferous epithelium, and (4) disorganization of the seminiferous epithelium. Scale bar: 100 µm.

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TABLE 3. Reproductive Organs Weight (g) at PND53 and PND90 in Male Rats Exposed to Silver Nanoparticles (AgNP) in PrepubertalPeriod

PND53, AgNP (µg/kg) PND90, AgNP (µg/kg)

Organ Control 15 50 Control 15 50

Body weight 253.4 ± 3.8a 242.1 ± 5.6a 226.4 ± 9.4b 380.1 ± 5.9 358.9 ± 8.5 377.5 ± 14.3Testis, caput 1.32 ± 0.03 1.26 ± 0.03 1.30 ± 0.07 1.61 ± 0.02 1.68 ± 0.06 1.59 ± 0.03Testis, corpus 0.12 ± 0.01 0.13 ± 0.01 0.13 ± 0.01 0.29 ± 0.01 0.32 ± 0.02 0.3 ± 0.03Testis, cauda 0.03 ± 0.01 0.02 ± 0.01 0.02 ± 0.01 0.04 ± 0.01 0.04 ± 0.01 0.04 ± 0.01Epididymis 0.07 ± 0.01a 0.06 ± 0.01b 0.06 ± 0.01b 0.23 ± 0.01 0.24 ± 0.01 0.23 ± 0.01Seminal vesicle, undrained 0.44 ± 0.064 0.31 ± 0.04 0.33 ± 0.05 1.37 ± 0.09 1.15 ± 0.08 1.2 ± 0.09Seminal vesicle, drained 0.31 ± 0.04 0.24 ± 0.03 0.25 ± 0.03 0.64 ± 0.04 0.58 ± 0.02 0.63 ± 0.03Ventral prostate 0.14 ± 0.02 0.12 ± 0.01 0.15 ± 0.02 0.37 ± 0.01 0.41 ± 0.06 0.41 ± 0.02

Note. Data are mean ± SEM. Superscript letters a and b differ in rows within the same age: p < .05. ANOVA and post hoc TukeyHSD; AgNP, silver nanoparticles.

DISCUSSION

The aim of the present study was to inves-tigate the adverse effects on reproductive func-tions following prepubertal exposure to AgNPin male Wistar rats. The dose of 15 µg/kg BW is10,000-fold lower than the lowest-observable-adverse-effect level (LOAEL) and 5000-foldlower than the no-observable-adverse-effectlevel (NOAEL). The dose of 50 µg/kg of BWcorresponds to 4000- and 1000-fold lower thanthe LOAEL and NOAEL, respectively, observedin rats exposed to AgNP (56 nm) for 90 d(LOAEL = 125 mg/kg and NOAEL = 30 mg/kg)(Kim et al., 2010). It is worthwhile notingthat no apparent symptoms of argyria wereobserved in this study, although these symp-toms were previously described (Drake andHazelwood, 2005).

The final weight of the animals atPND90 did not change markedly in rela-tion to earlier time-points, but there was lessgrowth in the 50-µg/kg BW AgNP treatmentgroup from PND34 to PND53. Shahare andYashpal (2013) found that Swiss-albino malemice treated orally with AgNP (3 to 20 nm)for 21 d exhibited decreases in body weightand damaged epithelial cell microvilli as wellas intestinal glands, which might be associatedwith a reduction of the absorptive capacityof the intestinal epithelium. In addition, inSprague-Dawley rats exposed to AgNP (60 nm)for 28 d, an accumulation of AgNP in the lam-ina propria in the small and large intestines

was observed, with higher numbers of gobletcells resulting in more mucus materials in theintestinal lumen (Jeong et al., 2010). The dosesof AgNP used in the studies just cited werehigher than the doses used in our investigation;however, it is possible that some injury in theintestinal epithelium may have occurred dur-ing the treatment and was reversed after theexposure ceased at PND53, allowing the ani-mals to recover intestinal absorptive function inthe subsequent days.

Puberty is the transitional period betweenthe juvenile phase and the adult state in whichsexual maturation begins in the hypothalamic–pituitary–gonadal axis and culminates in thedevelopment of secondary sexual characteris-tics and reproductive capability (Stoker et al.,2000). The parameter used to determine theprogression of puberty is typically preputial sep-aration (Korenbrot et al., 1977), and AgNPexposure produced a delay of puberty in bothtreatment groups while the rat weight did notchange. The increase in testosterone produc-tion by Leydig cells in testis is known to beresponsible for the separation of the preputialskin and exposure of the glans of the penis(Korenbrot et al., 1977). However, prepubertalexposure to AgNP did not produce signifi-cant alterations in serum testosterone levels atPND53 or PND90. It is possible that anothermaturational event in the hypothalamic–pituitary–gonadal axis other than testosteronelevels may have been affected by AgNP.

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Many factors may interfere with spermato-genesis, particularly during the prepubertalperiod, because testicular and epididymaldevelopment makes the process more sensitiveto the action of endocrine disruptors (Skinnerand Anway, 2005). The spermatogenesis pro-cess was affected in both groups treated withAgNPs and at both ages. Decreased spermreserves in the epididymis and diminishedsperm transit time were noted at PND53.Further, a reduction in the total and dailysperm production was observed at PND90.In vitro studies of spermatogonial cells in miceshowed cytotoxic effects (Asare et al., 2012)and an inhibition of mitochondrial activity(Braydich-Stolle et al., 2005; Carlson et al.,2008; Hadrup et al., 2012) due to expo-sure to AgNP, which manifests as a reduc-tion in proliferation of spermatogonial stemcells (Braydich-Stolle et al., 2010). AgNP arebelieved to disturb signaling by the glialcell line-derived neurotrophic factor (GDNF),which results in a reduction in the self-renewalof mouse spermatogonial stem cells in vitro(Braydich-Stolle et al., 2010). The weight of theepididymis was also lowered, possibly due todeceased sperm production.

The epithelial height of the seminiferousepithelium is related to sperm production(Romano et al., 2010, 2012), but marked dif-ferences in epithelial height were not found inthis study. Despite the reduction in sperm pro-duction, no significant change was observedin the seminiferous epithelium. The presenceof cellular debris and germinal epithelial cellsin the tubular lumen and vesicles is sugges-tive of impairment in the spermatogenesis pro-cess (Meistrich and Hess, 2013; Hess, 1990).In this situation, the seminiferous epithelium isoccupied by defective cells rather than sper-matozoa during development. In addition, itappears that a large number of spermatogonialnuclei are in the process of apoptosis; however,spermatogonial apoptosis occurs spontaneouslyin the seminiferous epithelium and thus maynot be a contributing factor (Allan et al., 1992).

In conclusion, prepubertal exposure toAgNP adversely affected reproductive devel-opment of prepubertal male Wistar rats. This

effect was reflected as impairment of the sper-matogenesis process and lower sperm countin adulthood. Evidence indicates that theseeffects were not reversed after the end of thetreatment.

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