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Reproductive Toxicology 44 (2014) 52–62

Contents lists available at ScienceDirect

Reproductive Toxicology

j ourna l ho me pa g e: www.elsev ier .com/ locate / reprotox

arbamazepine-exposure during gestation and lactation affectsubertal onset and spermatic parameters in male pubertal offspring

hayza Roberta Andretta1, Fatima Kazue Okada1, Camila Cicconi Paccola1,aiza Stumpp1, Samara Urban de Oliva ∗, Sandra M. Miraglia1

aboratory of Developmental Biology, Department of Morphology and Genetics, Federal University of Sao Paulo (UNIFESP), Rua Botucatu, 740, Ed. Lei tãoa Cunha, 2◦ andar, CEP: 04023-900 Sao Paulo, SP, Brazil

r t i c l e i n f o

rticle history:eceived 30 April 2013eceived in revised form4 September 2013ccepted 25 September 2013vailable online 12 October 2013

eywords:arbamazepine

a b s t r a c t

Carbamazepine (CBZ) is an anti-epileptic drug that acts on Leydig cells, affecting steroidogenesis andcauses fetal malformation. The aim of this study was to investigate the effects of CBZ on male sexual mat-uration and other male parameters. Rat dams were treated with CBZ during pregnancy and breastfeeding.The anogenital distance (AGD) and the anogenital index (AGI) were obtained. Testicular descent andpreputial separation were also evaluated. The offspring was euthanized at PND 41 and 63. The accessoryglands were weighed and the testes were collected for histopathological, morphometric and sterologi-cal analyses. The numerical density of Leydig cells and hormone dosage were obtained. CBZ caused anincrease of AGI and a delay of testicular descent and of preputial separation. CBZ also caused a decrease

estosteroneubertyperm parametersatregnancyreastfeedingeydig cell

of testosterone level and of sperm count and an increase of abnormal sperm. These results indicate thatCBZ delays puberty onset and affects steroidogenesis and sperm quality.

© 2013 Elsevier Inc. All rights reserved.

. Introduction

Antiepileptic drugs (AEDs) are used to treat a variety of neu-opsychiatric illnesses commonly encountered in women duringheir reproductive years, including epilepsy and bipolar disorder.or the past few decades, carbamazepine (CBZ) has been used as anffective treatment of seizures, bipolar disorder and certain types ofain [1]. Despite their widespread use, the impact of maternal expo-ure on fetal development remains obscure [2]. CBZ is one of theost commonly used antiepileptic drugs in Europe among women

f childbearing age [3]. Treatment of active epilepsy is importanturing pregnancy because seizures can lead to falls, injury andhysical stress that can endanger the health of the woman and theetus [4].

Various clinical and experimental studies involving the effectsf CBZ and other AEDs on male reproduction have been carriedut. However, they have focused mainly the seminal alterations

∗ Corresponding author. Tel.: +55 11 55764262; fax: +55 11 55764262.E-mail addresses: samaraurban@gmail.com (S.U. de Oliva),

iraglia.sm@gmail.com (S.M. Miraglia).1 Tel.: +55 11 55764262; fax: +55 11 55764262.

890-6238/$ – see front matter © 2013 Elsevier Inc. All rights reserved.ttp://dx.doi.org/10.1016/j.reprotox.2013.09.009

occurred as a result of drug administration in adult phase. Thealterations reported include reduced sperm motility and concen-tration, as well as sperm morphological alterations [5,6]. Anothercommon side effect related to the CBZ long-term treatment is anincrease of sex hormone binding globulin (SHBG) levels in theplasma, resulting in a reduction in the level of free bioactive testos-terone [7,8]. Moreover, CBZ can alter steroidogenesis by inhibitingthe cytochrome P450 monooxigenase system that is responsible forthe biosynthesis of the sexual steroid hormones [9,10]. Postnataltherapy with CBZ can lead to endocrine changes and has a nega-tive late impact on pubertal development and fertility of both boysand young men [11]. CBZ also provokes seminal alterations whenused during the adulthood [5,6]. In a previous study, our groupevaluated the side effects of CBZ on the spermatogenic process ofrats from weaning to peripuberty, puberty, as well as their sexualmaturation in adult phase; late seminiferous epithelium damageand alterations of the sex hormone levels were noted in these rats.CBZ, when administered from pre-puberty, can provoke specificside effects on rat testes, resulting in more severe damage in the

adult phase [12].

In addition, CBZ is able to cross membranes in the body such asblood–brain [13] and placental barriers [14,15]. It has been shownthat it can accumulate in the placental tissue and quickly reach the

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mbryo, leading to toxicity in post-implantation rat embryo [16].n addition, CBZ is also excreted into breast milk [15]. Althoughround 3000 pregnancies with recorded CBZ exposure have beenescribed in the literature, each individual study on its own isoo small to have the statistical power to detect risks for spe-ific congenital malformations compared with other antiepilepticrugs [3,17–19].

As previously mentioned, the effects of post-natal CBZ admin-stration on spermatogenesis have been addressed [5,6,12].onversely, experimental data about spermatogenesis and sexormone levels in rats exposed to the CBZ during the prenatalhase and breastfeeding were not found in the literature. This

s an important subject, since several studies showed that CBZxposure in the first trimester of pregnancy increases the riskor major congenital malformations [3]. It is also important toake into account that epileptic patients need to make contin-ous use of anticonvulsants and that, in pregnant women, therug withdrawal involves a balance of risks with loss of seizureontrol having potential implications not only for the mothernd the course of her pregnancy but, probable, her child as well20,21].

Considering all these data, we decided to scrutinize the effectsf CBZ treatment on the sex hormones and LH levels, as well asn testicular, epididymal and spermatic parameters in rats fromothers exposed to CBZ during all pregnancy and breastfeeding. In

ddition, we hypothesize that, in these circumstances, CBZ couldlter the adult Leydig cell population as well as act on the mas-ulinization programming window, delaying the puberty onset inhe progenies.

. Material and methods

.1. Animals

Female and male Wistar rats were housed in polypropyleneages (40 cm × 30 cm × 15 cm) filled with a layer of white pinehavings, under controlled conditions: hygiene, photoperiod (12 hight/dark cycle), humidity (60%) and temperature (22–23 ◦C). Theyad free access to tap water and commercial lab chow (Nuvilab,uvital Nutrientes). The females were mated overnight with males

two females per male); every morning, males were separated fromhe females and vaginal smears of each female were examinedor the presence of sperm; sperm presence in vaginal wet smearsas defined as the first day of pregnancy [22]. The rat dams were

reated with CBZ or propylene glycol during whole gestation andreastfeeding period, as sequentially described. Pregnant rats werebserved every morning for signs of toxicity. The pregnant ratsere housed individually and observed daily for delivery. The ratams were daily weighed during whole pregnancy and breastfeed-

ng for analysis of weight gain. From the progenies obtained, sixewborn rats (preferentially males) were kept with their damshroughout the breastfeeding period to obtain better and equaleeding for all pups. After weaning (21 days), the rats were main-ained in the cages (four per cage) at standard controlled conditions.he rats were submitted to euthanasia at 41 (peripuberty [23]) and3 (late puberty) days of age [24]. In rats, at 63 days of age, theestes are still growing and the number of step 19 spermatids perestis and per gram of testis and the daily sperm production areot stabilized [25]. The day of birth was considered postnatal dayPND) zero.

The experimental protocol followed the ethical principles

dopted by the Brazilian College of Animal Experimentation.he schedule for animal care and treatment was approvedy the local Institutional Ethics Committee (Protocol number513/11).

Toxicology 44 (2014) 52–62 53

2.2. Experimental schedules

A total of 48 male pups from Control and CBZ rat dams were usedin this study. Thus, the male progenies were randomly divided intotwo groups with 24 animals each: CBZ group, whose dams were20 mg/kg/day CBZ-treated (C-8981, Sigma Chemical Co., St. Louis,MO; 99.5% purity) diluted in propylene glycol (20 mg/mL) andControl (C) group, whose dams were treated with propylene gly-col (vehicle of CBZ; 99.8% purity; density 1.034 g/mL; 1 g/kg b.w.),following the same protocol of CBZ group. CBZ and propylene gly-col were administered via intraperitoneal route (i.p.) during allpregnancy and breastfeeding. The rats of each group were againdistributed into two subgroups (n = 12) according to the euthana-sia ages (PND 41 and 63). The CBZ dose chosen in the current studyis the usual anticonvulsive dose used for preventing kindled seizurein Wistar rats [26,27].

2.3. Body weight, anogenital distance and sexual development

At the PND 4, the body weight was obtained and the anogen-ital distance (AGD) of each male pup was recorded with a digitalmicrometer caliper. The AGD is defined as the distance betweenthe anterior end of the anus and the posterior end of the genitalpapilla [28]. The anogenital index (AGI) of male pups was also cal-culated; it is defined as the ratio between the AGD and the bodyweight at the moment of examination [AGI (mm/g) = AGD/bodyweight] [29]. After the obtainment of these morphometric mea-surements, the neonate male rats were kept with their damsuntil the weaning after what they were allocated in the cages (4rats per cage) up to the euthanasia ages: 41 or 63 days. Thus,the following subgroups (n = 12 each one) were formed based onthe type of their dam treatment and on the euthanasia age: (a)Control subgroups: C41 and C63, (b) CBZ subgroups: CBZ41 andCBZ63.

The weights of the male progenies (subgroups C41, CBZ41, C63and CBZ 63) were daily obtained, during the whole experiment toevaluate body weight gain. Each subgroup was constituted of ratsfrom different dams.

For sexual development evaluation, the day of the testiculardescent was monitored by daily palpation of the scrotum fromPND 15 [30]. From PND 33, the preputial separation was also dailyinvestigated through manual retraction of the prepuce [31–33]. Amagnifying glass was used for this goal. The preputial separationwas classified as: (a) stage 1: start of separation; (b) stage 2: prepucecould be retracted about halfway between the point of initial sepa-ration and the base of the phallus, and (c) stage 3: when separationwas complete [34].

2.4. Blood collection and hormonal analysis

The peripubertal and late pubertal rats were weighed andsubmitted to euthanasia through CO2 inhalation [35]. Clexane(Sanofi Winthrop Industry – France) (1 mL/kg) was administered10 min before the euthanasia. The blood was collected from theinferior cava vein and the plasma was separated and stored at−20 ◦C for further hormonal analyses [12]. Luteinizing hormone(LH), testosterone and estradiol plasma levels were determined byEnzyme-Linked Immunosorbent Assay (ELISA) using the kits (UscnLife Science Inc., Wuhan, P.R. China) according to the manufac-

turer’s instruction. The detection limit for LH was <144.5 pg/mL. Thedetection limits for testosterone and estradiol were 43.7 pg/mL and4.38 pg/mL, and the intra-assay variations were 1.8% for estradiol,and 4.5% for testosterone.

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.5. Morphometric and histological procedures

The PND 41 and 63 rats had their testes, epididymides, seminalesicle and ventral prostate removed and weighed. Full seminalesicles were weighed. The relative weight of the testis, epi-idymis, seminal vesicle and ventral prostate (mg/100 g of bodyeight) was also calculated. The testicular volume (Vt) of these

nimals was determined according to the Scherle’s method [36].n sequence, testes of PND 41 and PND 63 rats were immersion-xed in Bouin’s liquid according to Russell et al. [37] and Noltet al. [38]. One half of each collected testis was fixed for 48 h andaraplast Plus-embedding (P-3683, Sigma Chemical Co., St. Louis,O) for histopathological, morphometric and stereological analy-

es. The other testicular half was fixed for 24 h and embedded inaraffin for immunohystochemical study (item 2.9). At the PND 63,nly the left testis was collected for histopathological, morphomet-ic, stereological and imunohistochemical analyses while at PND 41he both testes were used for this goal; the right testes of PND 63ats were frozen for subsequent spermatic analysis.

.6. Morphometric and stereological testicular analyses

Four micrometer-thick testicular cross-sections from the 48 h-xed Paraplast blocks were submitted to the Periodic Acid-Schiffistochemical method with Harris’s Hematoxylin counterstainingPAS + H) [39,40]. The seminiferous tubule sections were systemat-cally randomly sampled and scrutinized using the Leica QWin-V3Cambridge, UK) image analysis system, using 20× and 40× objec-ive lenses; the images were captured using a digital cameraonnected to a light microscope.

Volume densities (Vv) of the seminiferous epithelium, lymphaticpace, interstitial tissue and tubular lumen were achieved at PND1 and 63 using a 25-point integrating eyepiece attached to a lightinocular microscope [41]. The points were counted in 30 randomelds of testicular sections, totalizing 750 points per animal (forach section analyzed). The volume (V) of each component of theestis was estimated by multiplying the respective volume densityVv, in percentage) by the total testis volume (Vt, Scherle’s method36]) and dividing this result by 100 [42].

The diameter of the androgen-dependent tubule sections [43],.e., stages VII and VIII at PND 41 and 63, was also determinedsing the Leica QWin-V3 (Cambridge, UK) image analysis system.he testicular sections of CBZ and Control rats, in both ages cited,ere totally scrutinized and the seminiferous tubule sections in

ndrogen-dependent stages were measured using 20× objectiveens magnification. When the sections were slightly oblique, onlyhe minor axis was considered [42].

.7. Sperm count, daily sperm production and sperm transit time

The right testes and epididymides of PND 63 rats were collectedt the same time and frozen at −20 ◦C. The organs were thawednd homogenized. Homogenization-resistant testicular elongatedpermatid nuclei with a shape characteristic of step 17–19 sper-atids and sperms were respectively collected from the right testis

nd from the right epididymis (caput/corpus and cauda) of the Con-rol and CBZ-exposed animals at PND 63, for analysis and count,s previously described [25]. The testicular and epididymal spermumber were expressed by organ and in grams (g) of organ. Dailyperm production (DSP) was obtained dividing the total numberf homogenization-resistant spermatids per testis by 6.1 days, as

his is the length of time that spermatids are kept in the epithe-ium in each seminiferous cycle. The sperm transit time throughhe epididymis was determined dividing the sperm number in eachortion by the DSP [25].

Toxicology 44 (2014) 52–62

2.8. Sperm morphology

Samples of fluid were obtained from the cauda of the left epi-didymis of the PND 63 rats (subgroups CBZ63 and C63) for spermmorphological analysis. Samples of 3 �L of the epididymal fluidobtained from the left epididymis cauda were homogenized in2 mL of bidistilled water. One drop of the solution was smearedonto a glass slide and air-dried. The smears were stained by theShorr/hematoxylin method.

For morphological evaluation, 200 spermatozoa were ran-domly analyzed and the percentage of abnormal spermatozoa wasobtained. The abnormal characteristics considered were: (1) headabnormalities and (2) tail abnormalities. The presence of round cells[44] in the epididymis fluid was also investigated.

2.9. Imunohistochemical analysis

Testicular cross-sections (6 �m) of CBZ and Control groups at41 and 63 PND were obtained from the 24 h-fixed paraffin blocks.The sections were submitted to the immunolabeling of Leydigcells (LCs) for 11�-Hydroxysteroid Dehydrogenase type II. The sec-tions were deparaffinized, hydrated, treated with proteinase K(20 �g/mL) for antigen retrieval and submitted to endogenous per-oxidase inactivation (3% H2O2). The sections were treated with 0.1%Triton-X100 (30 min) and 20% BSA (1 h). The slides were incubatedwith the primary antibody anti-11�-Hydroxysteroid Dehydroge-nase type II (sheep anti-rat 11�-HSD-II – AB1296, Chemicon®,1:1500) at room temperature for 1 h. The slides were washed inPBS (0.05 M, pH 7.4) and incubated with the biotinilated secondaryantibody and streptavidin-HRP (LSAB kit, DAKO®) for 30 min each.The reaction was revealed by 3,3′-diaminobenzidine (DAB, DAKO®)and the sections were counterstained with Harris’s Hematoxylin.Only cells showing intense dark-brown labeling were consideredpositive. Negative controls were performed by omitting the pri-mary antibody. The enzyme 11�-HSD-II is present only in adult LClineage [45,46].

2.10. Numerical density (Nv) of Leydig cells (LCs)

To assess the relative numbers of 11�-HSD-II-positive LCs atPND 41 and 63, the numerical density (Nv) of these cells wasobtained. The Nv was expressed by the ratio between the num-ber of labeled cells and the volume of the tissue analyzed [47]. Thecell counting and the tissue volume were obtained using the LeicaQWin-V3 (Cambridge, UK) image analysis system, using 50× objec-tive lens magnification. The images were captured using a digitalcamera connected to a light microscope.

Thirty fields per rat were randomly analyzed in the intersti-tial tissue of each left or right testicular section, depending on theeuthanasia age. To avoid overestimation of cell counts due to sec-tion thickness, a unique focus plan (upper plan) was chosen [47,48].Only the cell profiles that showed a conspicuous and clear nucleuswere scored. To obtain the Nv, the volume of Optic Disector (Vdis)[49] was calculated according to the following formula: [Vdis = areaof the interstitial tissue examined × section thickness].

Thus, the total volume of Optical Disector (�Vdis) was calculatedas the sum of the areas corresponding to all interstitial testicularfields analyzed multiplied by the section thickness. As a result, Nv

of the LCs could be obtained dividing the total number of the scoredcells by the total volume of Disector:

[Nv = total number of scored LC/total volume of the analyzed

interstitial tissue].

Thus, the total number of 11�-HSD-II-positive LCs wasexpressed per centimeter cubic (cm3) of testicular interstitial tis-sue.

R.R. Andretta et al. / Reproductive Toxicology 44 (2014) 52–62 55

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Fig. 2. AGD (mm) from Control and in utero CBZ-exposed male pups (PND 4). Valuesexpressed as median and interquartile intervals. Mann–Whitney’s test. n = 12.

reduction compared to C41 rats (Fig. 4), whereas the averagediameter of seminiferous tubules at all stages of the seminiferousepithelium cycle as well as the diameter of the tubular sections at

Table 1Ages of testicular descent and body weight of the litters on the day of testiculardescent and preputial separation in Control and CBZ-exposed rats.

Parameters Control CBZ-exposed

Testicular descent(days)

18.4 ± 0.73 19.92 ± 0.80*

Weight (g) of the pupsat the day oftesticular descent

38.63 ± 5.4 41.19 ± 4.62

Preputial separation(days)

44.36 ± 1.84 46.09 ± 2.69*

Weight (g) of the pups 214.50 ± 11.56 193.91 ± 17.60*

ig. 1. Body weight gain in male pups from CBZ-exposed and Control dams in theeriod from PND 4 to PND 63. Values expressed as mean. Standard deviation is nothown. Student’s t-test. n = 12. 4 → 50 PND: *p ≤ 0.05. 51 → 63 PND: p > 0.05.

.11. Statistical analysis

The data were submitted to parametric or non-parametric testssing SigmaPlot software 12.0. Data that passed the normalityest were submitted to t-test (Student’s test). The non-parametric

ann–Whitney’s test was used to compare data that failed the nor-ality test. Differences were considered significant when p ≤ 0.05.

. Results

.1. Pregnancy length and body weight gain

The CBZ-exposed dams did not show significant alterations inody weight gain during pregnancy (body weight at the end ofregnancy (g) – C: 356.99 ± 61.06, CBZ: 353.57 ± 50.19) and in thereastfeeding period (body weight in the end of breastfeeding (g)

C: 278.82 ± 43.80, CBZ: 314.62 ± 40.52) when compared to theespective Control dams.

.2. Body weight gain of pups

The treatment of dams with CBZ during pregnancy and breast-eeding caused a significant decrease of the body weight gain inhe male offspring from PND 4 to PND 50. From the PND 51 toND 63 this parameter did not show significant alteration in theffspring from CBZ-exposed dams in comparison with the Controlam offspring (Fig. 1).

.3. Anogenital distance (AGD) and anogenital index (AGI)

Significant alterations of the AGD were not observed in maleups from the control and CBZ-treated dams (Fig. 2). On the otherand, the AGI showed a significant increase in male pups from CBZ-reated dams (Fig. 3).

.4. Descent of the testis and preputial separation

CBZ-exposed offspring presented significant delay of the timef the testicular descent and of preputial separation. Moreover, theelay in time of preputial separation coincided with a decrease ofody weight (Table 1). The preputial separation was completed inoth groups.

.5. Reproductive parameters in the peripuberty and late puberty

Male progenies from CBZ-treated dams during whole pregnancynd breastfeeding showed a decrease of the final body weight and

Fig. 3. AGI (mm/g of b.w.) of the male pups (PND 4) from Control and CBZ-exposeddams during pregnancy. Values expressed as mean ± SD. *p ≤ 0.05. Student’s t-test.n = 12.

of the absolute testicular and ventral prostate weights at PND 41(CBZ41 subgroup) when compared to the corresponding C41 Con-trol subgroup. Significant reduction in the ventral prostate relativeweight was also noted in animals of the CBZ63 subgroup in com-parison with the C63 Control subgroup. Nonetheless, the absoluteand relative weights of the epididymis and full seminal vesicle fromCBZ-exposed groups (CBZ41 and CBZ63) did not show significantdifferences in comparison with their Control subgroups (C41 andC63) (Table 2).

The testicular volume in the CBZ41 rats showed significant

at the day ofpreputial separation

Values expressed as mean ± SD. Student’s t-test. n = 12.* p ≤ 0.05.

56 R.R. Andretta et al. / Reproductive Toxicology 44 (2014) 52–62

Table 2Final body weights, absolute and relative epididymis, ventral prostate, seminal vesicle and testicular weights from Control (C41 and C63) and CBZ-exposed (CBZ41 andCBZ63) rats.

Peripuberty Late puberty

C41 CBZ41 C63 CBZ63

Final body weight (g) 195.73 ± 16.60 162.42 ± 25.35* 311.08 ± 23.69 293.61 ± 24.37

Absolute weights (mg)Testis 896.00 ± 66.42 770.00 ± 124.41* 1562.00 ± 157.00 1516.00 ± 117.00Epididymis 124.66 ± 18.07 109.28 ± 23.03 366.00 ± 35.50 348.00 ± 44.10Ventral prostate 102.67 ± 19.07 80.71 ± 17.74* 233.00 ± 36.10 249.00 ± 38.20Full seminal vesicle 63.07 ± 11.82 54.61 ± 11.28 672.31 ± 76.72 704.62 ± 157.09

Relative weights (mg/100 g)Testis 459.32 ± 35.05 474.95 ± 39.30 502.99 ± 44.76 519.62 ± 36.55Epididymis 63.88 ± 9.49 67.20 ± 10.08 117.79 ± 8.42 120.15 ± 12.51Ventral prostate 52.64 ± 9.85 50.65 ± 13.20 75.11 ± 11.31 85.74 ± 12.95*

Full seminal vesicle 35.07 ± 7.93 38.55 ± 9.41 216.71 ± 24.58 239.04 ± 46.75

Values expressed as mean ± SD. Student’s t-test. n = 12.* p ≤ 0.05.

Fig. 4. Testicular volume (cm3) in Control (C41 and C63) and CBZ-exposed (CBZ41and CBZ63) groups. Values expressed as mean ± SD. *p ≤ 0.05. Student’s t-test. n = 12.

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Fig. 5. Plasma testosterone levels (pg/mL) in Control and CBZ-exposed rats. Values

mal sperm frequency in the CBZ63 rats was observed (Table 4). The

TV(

V

ndrogen-dependent stages (VII and VIII) did not show significantifferences in the animals of both Control and CBZ groups at PND1 and 63 (data not shown).

The volume density (Vv) of the testicular components (seminif-rous epithelium, interstitial tissue, lymphatic space) and of theubular lumen of the CBZ41 and CBZ63 rats did not show sig-ificant alterations when compared to the Control subgroups atorresponding ages. Evident reductions in the volume (V) of theeminiferous epithelium and lymphatic space were observed in theBZ41 subgroup in comparison with the respective Control rats

Table 3).

able 3olume density (Vv) and total volume (V) of the seminiferous epithelium, interstitial ti

CBZ41 and CBZ63).

Stereological parameters Peripuberty

C41 CB

Vv seminiferous epithelium (%) 68.08 ± 4.92 6Vv interstitial tissue (%) 11.90 ± 2.54 1Vv lymphatic space (%) 9.02 ± 1.85

Vv lumen (%) 11.00 ± 2.44 1V seminiferous epithelium (mm3) 552.23 ± 44.26 44V interstitial tissue (mm3) 97.64 ± 17.14 9V lymphatic space (mm3) 85.51 ± 18.61 5V lumen (mm3) 88.86 ± 19.35 8

alues expressed as mean ± SD. Student’s t-test. n = 7.* p ≤ 0.05.

expressed as median and interquartile intervals. *p ≤ 0.05. Mann–Whitney’s test.n = 12.

3.6. Spermatic parameters

The exposure of dams to CBZ during gestation and breastfeed-ing caused a significant decrease in sperm count in the epididymiscauda in the offspring at PND 63 (CBZ63 subgroup). On the otherhand, no difference was observed in the counts of testicular step19 spermatids, daily sperm production, sperm count in epididymalcaput/corpus and sperm transit time between the Control (C63) andCBZ-exposed (CBZ63) subgroups (Table 4).

In addition, a significant increase of the morphologically abnor-

most frequent abnormality observed was the presence of isolatedsperm tails [Control: 4% (1.5–6.5), CBZ63: 11% (7.75–12.75); data

ssue, lymphatic space and lumen in Control (C41 and C63) and CBZ-exposed rats

Late puberty

Z41 C63 CBZ63

7.94 ± 0.81 59.15 ± 4.76 61.95 ± 1.712.96 ± 1.43 13.52 ± 2.72 12.26 ± 2.928.15 ± 1.04 14.73 ± 2.60 13.31 ± 3.300.95 ± 2.00 12.60 ± 1.41 12.48 ± 1.335.93 ± 93.94* 840.83 ± 66.04 890.60 ± 63.042.13 ± 22.63 221.72 ± 49.27 171.62 ± 41.512.54 ± 5.23* 241.60 ± 52.60 215.89 ± 47.613.87 ± 18.19 183.63 ± 32.67 188.56 ± 28.47

R.R. Andretta et al. / Reproductive Toxicology 44 (2014) 52–62 57

Table 4Testicular and epididymal spermatic parameters in the C63 and CBZ63 subgroups.

Parameter C63 CBZ63

TestisSpermatid number(106/testis)a

109.06 ± 22.63 108.43 ± 27.10

Spermatid number(106/g/testis)a

91.72 ± 18.98 85.92 ± 21.47

Daily sperm production(106/g/day)a

17.88 ± 3.72 17.77 ± 4.55

Epididymal caput/corpusSperm number (106/organ)a 52.82 ± 11.68 51.28 ± 12.68Sperm number(106/g/organ)a

325.38 ± 50.65 294.02 ± 43.19

Sperm transit time (days)b 2.85 (2.29–3.60) 2.62 (2.07–3.63)

Epididymal caudaSperm number (106/organ)a 57.31 ± 15.84 43.54 ± 9.72*

Sperm number(106/g/organ)a

557.38 ± 89.12 428.86 ± 104.93*

Sperm transit time (days)b 2.49 (2.21–3.74) 2.08 (1.62–3.13)

Sperm morphologyb

Normal (%) 94.80 ± 0.97 91.37 ± 1.93Abnormal (%) 5.20 ± 0.97 8.63 ± 1.93*

a Values expressed as mean ± SD. Student’s t-test. n = 12.b Values expressed as median and interquartile intervals. Mann–Whitney’ test.

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xpressed in median and inter-quartile range; p ≤ 0.05]. Round cellsere not observed in the epididymal fluid of the C63 and CBZ63 rats.

.7. Hormone levels

Plasma testosterone level was significantly lower in CBZ41 ratsn comparison with the C41 Control rats (Fig. 5). The estradiol andH plasma levels did not show significant alterations when CBZ ratsnd their corresponding Control rats were compared (Figs. 6 and 7).

.8. Numerical densities of 11ˇ-HSD-II-positive cells

The presence of 11�-HSD-II enzyme was detected in Leydig cellLC) cytoplasm in all subgroups (Fig. 8). The numerical density of1 �-HSD-II-positive LCs was not statistically different when CBZ41

nd CBZ63 subgroups and their respective Control subgroups wereompared among them (Fig. 9).

ig. 6. Plasma estradiol levels (pg/mL) in Control and CBZ-exposed rats. Valuesxpressed as median and interquartile intervals. Mann–Whitney’s test. n = 12.

Fig. 7. Plasma LH levels (pg/mL) in Control and CBZ-exposed rats. Values expressedas median and interquartile intervals. Mann–Whitney’ test. n = 12.

3.9. Histopathological analysis

Rat testicular sections of the C41 and C63 Control subgroups,as well as of the CBZ41 and CBZ63 subgroups showed normalhistological characteristics in the different phases of sexual devel-opment and also presented normal and typical cellular associationsof the stages of the seminiferous epithelium cycle (Fig. 10).

4. Discussion

The reproductive hormone status in humans is considerablydifferent among adults, children and adolescents. Endocrine alter-ations due to long-term CBZ treatment may cause a negative effecton pubertal development and fertility of boys and young men [11].The repercussion of drug action on puberty onset can be even morecomplex when the treatment occurs during pregnancy and contin-ues during breastfeeding. Anogenital distance, preputial separationand testicular descent are androgen dependent phenomena thatindicate pubertal development and are important parameters to beconsidered when scrutinizing the anti-androgen effects of a givenagent.

AGD measurement is an indicator for endocrine disruption inanimals and may be shorter in infant males with genital anomalies[50]; this measurement has been used, for example, as a sensitivebiomarker of the effects of antiandrogens such as dibutylphtha-late [51], flutamide [52], finasteride [53] and atrazine [54]. Besides,longer anogenital distance may predict normal male reproductivepotential [50]. In addition, the anogenital index (AGI) reflects themasculinization process and is significantly higher in males than infemales [55]. In the rat, it has been demonstrated that androgen-mediated development of normal male reproductive system occursvia androgen “programming” within a specific fetal time window[embryonic days 15.5–18.5], that precedes morphological differ-entiation and relevant development of the tissues [56]. So, reducedanogenital distance (AGD) can be used to scrutinize the androgenproduction/action during the masculinization programming win-dow (MPW). In the present study, considering that AGD did not varyin male pups, it is possible to suggest that the exposure of rats to CBZduring the whole pregnancy did not have a relevant action in theMPW. Conceptually, it is reasonable to anticipate that AGD mightvary with pup body weight. Therefore, agents with no endocrineactivity might induce apparent AGD alterations if they affect over-

all pup size. Conversely, AGD alterations might go undetected afterthe treatment with agents that have hormonal effects if they alsoinduce an offset change in pup body weight [57]. As previouslymentioned, a decrease of body weight in the CBZ-exposed male

58 R.R. Andretta et al. / Reproductive Toxicology 44 (2014) 52–62

Fig. 8. Photomicrographs of testicular sections showing 11�-HSD-II-positive Leydig cells (LCs) (Brown-stained adult LC cytoplasm) in PND 41 (A and B) and PND 63 rats (C andD 14 �ma

pi

iolci

FCS

) pertaining to the Control (A and C) and CBZ (B and D) groups. Bars of A–D: 11 �m,

dult LC. Bar: 6 �m.

ups occurred and indeed, this event contributed to the significantncrease of AGI.

Alterations of body weight have been commonly observed dur-ng treatment with antiepileptic drugs. A transient retardation

f early postnatal growth (lower body weight and smaller meanength increment in the first postnatal month) has been noted inhildren of epileptic mothers exposed to CBZ in utero in compar-son to non drug-exposed children, what could possibly involve a

ig. 9. Numerical densities (Nv) of 11�-HSD-II-positive LCs from control (C41 and63) and CBZ-exposed (CBZ41 and CBZ63) groups. Values expressed as mean ± SD.tudent’s t-test. n = 5.

, 12 �m and 11 �m, respectively. Observe, in the inset detail, a 11�-HSD-II-positive

reversible suppression of thyroid function [58]. However, in thepresent study, the decrease in the body weight gain caused by CBZlasted from birth until PND 50. The influence of CBZ in the bodyweight seems to be controversial since, in a previous study, ourgroup showed that rats treated with CBZ from the weaning untilthe peripuberty or puberty (respectively PND 43 and 63) did notshow a significant alteration of body weight at euthanasia day [12]or significant alterations of the body weight gain during the exper-iment (unpublished data). This difference between our present andthe previous results is probably related to the different periods oftreatment.

No difference in the numerical density of adult Leydig cells wasobserved between Control and CBZ rats at the ages investigated(PND 41 and 63). These data were unexpected since, in these rats,the significant testosterone level reduction at PND 41 indicatessteroidogenesis alteration. This last datum corroborates with theinterference of CBZ with the puberty onset. In fact, plasma andtesticular testosterone levels have the first post-natal significantincrease between days 39 and 41 [59,60] after this peak, testos-terone levels increase progressively up to 50 days of age [59]. Thus,although testosterone level was not daily measured in the presentstudy, it is reasonable to consider the possibility that testosteronereduction observed at PND 41 was maintained until PND 50, whenthe rat becomes pubertal [25], but this needs confirmation. Indeed,

puberty is a time of significant weight gain and so, during pubertaldevelopment, interactions between growth hormone (GH) and thesex steroid hormones are striking and pervasive. Studies in adoles-cent boys showed that the rising concentrations of testosterone

R.R. Andretta et al. / Reproductive Toxicology 44 (2014) 52–62 59

Fig. 10. Photomicrographs of testicular sections of PND 41 (A–D) and PND 63 (E–H) rats submitted to the periodic acid-Schiff histochemical method and counterstained withHarris’ Hematoxylin (PAS + H). (A–H) Note the normal morphology of the seminiferous epithelium showing typical germ cell associations in Control subgroups (A, B, E andF) and CBZ subgroups (C, D, G and H). Bars: 47 �m, 17 �m, 47 �m, 17 �m, 47 �m, 17 �m, 24 �m and 9 �m, respectively.

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uring puberty play a pivotal role in augmenting spontaneousecretion of GH and production of insulin-like growth factor I (IGF-) [61]. Therefore, a possible slower increase of testosterone levelould be considered as an adding factor to the concomitant reduc-ion of the body weight gain in CBZ rats.

Besides altering sex hormones, CBZ treatment can also affectther parameters that control the synthesis of these hormones,uch as the peptide ghrelin, a potent stimulator of growthormone secretion from the anterior pituitary gland. Ghrelin dose-ependently inhibits the testicular testosterone secretion in vitro,odulates Leydig cell proliferation in vivo [62] and regulates

ey aspects of testis physiology, such as steroidogenesis, Leydigell proliferation and tubular function [63]. Levels of ghrelin areeduced in epileptic children under treatment with CBZ in compar-son to healthy age- and weight-matched subjects [64]. Consistent

ith its role in modulating gonadotropin secretion, ghrelin delaysubertal onset, in both male and female rats, but males appear toe more sensitive than females [63]. Thus, it is possible that, in theresent study, the in utero and breastfeeding exposure of malesas caused an alteration of ghrelin level that could, indirectly, con-ribute to the reduction of testosterone level at PND 41 and lead tolterations of other androgen-dependent events, such as balano-reputial separation (BPS) and testis descent delay observed in CBZats. In rodents, the testicular descent occurs after PND 15 [30] andhe BPS, an external sign of pubertal development in the male rat,ccurs around PND 39 [65]. It is has been suggested that elevatedhrelin levels may operate as a negative modifier of key reproduc-ive states, such as pregnancy and male puberty onset and partiallyrevents the normal occurrence of BPS [66].

Testosterone has an important role in the development of theale accessory glands. In the rat, these glands grow from around

3–49 days of age until around 70 days [67,68]. Indeed, in theresent study there was a decrease of the ventral prostate weightt PND 41 and of its relative weight at PND 63, corroborating withhe reduction of testosterone level at PND 41. Reductions of testos-erone level [12] and of ventral prostate weight [69] were alsobserved in PND 93 adult rats treated with CBZ from weaning untildulthood. Additional studies are needed to investigate the sexormone profile in the adult male offspring from CBZ-exposed ratams. Conversely, seminal vesicle weight and androgen-dependentubule diameter did not differ among CBZ and Control rats asxpected. The prostate is more sensitive to the testosterone actionuring the pre-puberty and adulthood than at puberty whereashe seminal vesicle of rats shows major sensitivity during there-puberty [70–72]. This could explain the absence of significanteight alterations of the seminal vesicle.

As aforementioned, although the plasma testosterone level waseduced at PND 41, significant alterations in androgen-dependentubule diameters of PND 41 and 63 CBZ rats were not observed.owever, significant decrease of testicular weight and volumeccurred at 41 day-old CBZ-exposed rats when compared to Controlats. Testicular weight reduction was also observed by our groupn a previous study [12] at PND 43, after treatment with CBZ inhe post-natal phase. In addition, in that study, conspicuous histo-ogical alterations of the seminiferous epithelium were described.n the present study, as the histological seminiferous epitheliumhanges were not observed in the CBZ rats at both ages studied, theecrease of seminiferous epithelium volume could be explainedy the slower testicular growth as a result of the delay of pubertyaused by CBZ. The significant decrease of lymphatic space volumen PND 41 CBZ-exposed rats could also be a result of the pubertyelay and testicular volume reduction. In addition, testicular inter-

titial fluid volume can also change due to a variety of factors,ncluding testosterone level alterations [73] and toxic exposure74–76]. Indeed, the reduction of lymphatic space observed heregrees with the testosterone level reduction.

Toxicology 44 (2014) 52–62

Although CBZ-exposed rats did not show testicular histologicalalterations, there was a reduction in the sperm concentration in theepididymal cauda at PND 63. The concept that the epididymis actsas a quality-control organ to remove defective spermatozoa beforeejaculation has been proposed by Sutovsky et al. [77] based onsperm ubiquitination. Defective mammalian spermatozoa wouldbe ubiquitinated during epididymal passage, a mechanism thatmay mark the abnormal spermatozoa for proteolytic destruction.According to these authors, part of the ubiquitinated sperms wouldbe phagocytozed prior to reaching the epididymis cauda [77]. A sig-nificant increase in the number of abnormal sperm occurred in theCBZ-exposed rats at PND 63 in comparison to Control rats. Thus,sperm phagocytosis in the epididymis could partially explain thereduction of sperm count in the epididymis cauda observed in theCBZ rats at PND 63. However, this issue should be carefully inves-tigated in future studies. Another possibility is that a significantnumber of these anomalous sperms died before they had arrivedthe epididymis and were phagocytozed by the Sertoli cells [78],event that normally takes place in the distal region of deferent vas[79]. On the other hand, a more plausible reason to explain thereduction of the sperm concentration in the epididymis cauda atPND 63 would be the reduction of the sperm transit time, but thislast parameter did not show significant alteration in CBZ rats incomparison to Control rats.

In conclusion, the data presented here (later preputial sepa-ration and testicular descent, reduction of testosterone level atPND 41, reduction of weight and testicular volume and of ven-tral prostate weight) indicate that CBZ treatment during pregnancyand breastfeeding caused a delay of puberty onset and spermalterations in male progenies at late puberty. This is an impor-tant issue that deserves additional investigation. However, someaspects, such as the reduction of the weight in the neonate pupsand CBZ interference in steroidogenesis, need to be clarified, sincean anti-androgenic effect of CBZ during fetal phase cannot be dis-carded. In addition, it is possible that other paracrine and endocrinemechanisms, such as ghrelin and thyroid hormone levels, can alsobe involved in the reproductive alterations observed, indicating amore complex action of CBZ on reproduction.

Conflict of interest statement

The authors declare that there are no conflicts of interest.

Acknowledgments

The authors thank Prof. Helena B. Nader (Department of Phar-macology EPM/UNIFESP) for helping in the testosterone analysisand CAPES (Coordenac ão de Aperfeic oamento de Pessoal de NívelSuperior) and FAPESP (Fundac ão de Amparo a Pesquisa do Estadode São Paulo - Processo 2012/05905-9) for financial support.

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