Sperm impairments in adult vesper mice (Calomys laucha) caused by in utero exposure to bisphenol A

ORIGINAL ARTICLE

Sperm impairments in adult vesper mice (Calomys laucha)caused by in utero exposure to bisphenol AJ. Vilela1, A. Hartmann1, E. F. Silva2, T. Cardoso1, C. D. Corcini1,2, A. S. Varela-Junior1, P. E. Martinez1

& E. P. Colares1

1 Instituto de Cie^ncias Biologicas, Universidade Federal do Rio Grande (FURG), Rio Grande, RS, Brazil;

2 ReproPel, Faculdade de Veterinaria, Campus Cap~ao do Le~ao, Universidade Federal de Pelotas (UFPel), Pelotas, RS, Brazil

Keywords

BisphenolCalomysendocrine disruptor

reproductionsemen

Correspondence

Janice Vilela, Programa de Pos-Graduac~aoem Cie^ncias Fisiologicas: Fisiologia Animal

Comparada - Instituto de Cie^ncias Biologicas,

Universidade Federal de Rio Grande. Av. Italia

km 8 Bairro Carreiros, Rio Grande,

RS 96203-900, Brazil.

Tel.: +55 (61) 8305 0527;

Fax: +55 (53) 3233 6848;

E-mail: [email protected]

Accepted: September 9, 2013

doi: 10.1111/and.12182

Summary

This study aimed to evaluate the effects of in utero administration of bisphenol A

(BPA) on semen parameters of vesper mice. Sixty female Calomys laucha were

divided into six groups and received by gavage during gestation the following sub-

stances: Water (negative control), Olive Oil (vehicle control), Diethylstilbestrol

(DES positive control 6.5 lg kg1 bw) and BPA (40, 80 and 200 lg kg1 bw).Male offspring were euthanised at 70 days of age, and sperm parameters were anal-

ysed. BPA reduced normal sperm morphology (water = 96.1 0.65;BPA200 = 96.8 2.3%), sperm membrane integrity (water = 88.8 1,65; BPA200 = 70.6 4,15%), sperm motility (water = 87.5 1.71; BPA200 = 51.3 9.9%) and in vitro penetration rates (water = 55.0 7.14; BPA200 = 7.47 2.96%), but it did not affect body weight, anogenital distance, sperm DNA integ-

rity and acrosome integrity. In conclusion, in utero exposure to BPA caused a

reduction in sperm parameters of adult C. laucha. Natural mating studies should

be conducted to verify the effects of BPA on fertility of the animals.

Introduction

It is well documented that several natural and man-made

chemicals interfere with the hormonal system of vertebrate

and invertebrate organisms (Pawlowski et al., 2004), acting

as endocrine disruptors both directly, by binding to

hormonal receptors, and indirectly, by modulating endoge-

nous hormone levels, by interfering with biochemical

processes associated with the production, availability or

metabolism of hormones, or also by the modulation of

receptors (Lagadic et al., 2007). One of them is bisphenol

A (BPA), a substance used in the production of

polycarbonate plastic and epoxy resins which are used in

manufacturing plastic containers, baby bottles and other

products (Geens et al., 2012). This chemical was found to

be an important endocrine disruptor that acts by mimick-

ing estrogens (Welshons et al., 2006), and it has been

detected in air, fresh and marine waters, soil and sediments

(Rodriguez-Mozaz et al., 2005; Vandenberg et al., 2007; Fu

& Kawamura, 2010; Flint et al., 2012; Huang et al., 2012).

Many studies have shown that mice perinatally exposed

to BPA present impaired fertility (Toyama & Yuasa, 2004;

Salian et al., 2009, 2011; LaRocca et al., 2011) as well as

increased anogenital distance and reduced prostate size

and epididymis weight (Gupta, 2000). Also, BPA has been

reported reducing sperm count in rats that ingested 25

and 100 ng kg1 (Al-Hiyasat et al., 2002); in humans,higher doses of urine BPA were related to lower sperm

concentration and motility and higher sperm DNA

damage (Meeker et al., 2010; Li et al., 2011).

While most of the studies of BPA effects aim to

extrapolate the results to human impacts, there are few

studies concerning its effects on wildlife (Nieminen et al.,

2002a,b). Evaluation of BPA exposure on mammals cur-

rently relies on data from laboratory studies on model

animals, such as mice and rats, and indicates many detri-

mental effects on rodents at high levels of BPA. However,

wildlife species may be exposed to chemicals at low doses

and thus act as bioindicators for the effects of endocrine

disruptors in natural ecosystems (Flint et al., 2012). The

vesper mouse (Calomys laucha, Waterhouse 1837) is a

small rodent found in South America (Reis et al., 2006)

and it is of epidemiological relevance as a reservoir of

Hantavirus and of protozoa pathogenic to humans (Mills

2013 Blackwell Verlag GmbH 1Andrologia 2013, xx, 18

et al., 1994). Its spermatozoon has a rounded head,

similar to that of no rodent mammals, and their small

size and rapid reproductive cycle are beneficial to experi-

mental studies (Lasserre et al., 2000). Moreover, this

species has some advantages over conventional rodents

for toxicological studies because of its genetic variability

and resistance to stressful agents (Vandenbergh, 2004).

While the main source for human exposure to BPA is

food and liquid storage containers, BPA is also released

into the environment through sewage treatment effluent

(via human-ingested BPA being eliminated through

sewage), landfill leachate (via hydrolysis of BPA from

plastics) and natural degradation of polycarbonate plastics

(Crain et al., 2007). As vesper mouse lives on coasts, in

pastures and along roadsides (Reis et al., 2006), this spe-

cies may be affected by environmental sources of BPA.

Since these animals still present wild characteristics, what

is observed in captivity may be similar to what happens

in wildlife. Therefore, this study aimed to verify the

effects of in utero exposure to BPA on sperm parameters

of adult Calomys laucha.

Materials and methods

Animal handling

Animals were from the vivarium of nonconventional

animals at the Federal University of Rio Grande (FURG),

Rio Grande do Sul, Brazil. Animal collection and

maintenance in captivity were approved by the Brazilian

Institute for Environmental Protection (IBAMA, permit

#14174-1). Animals used in this study were from F5 gen-

eration kept in the vivarium. Parental crosses were from

selected animals so as to prevent brothersister matings.The animals were individually housed in plastic boxes

(35 9 20 9 13 cm), kept in an environment with

controlled temperature (20.0 2.0 C) and photoperiod(LD 12:12, with light on at 6:00 a.m. and off at 6:00

p.m.), and received soy-free diet and water ad libitum.

Experimental design

Sixty female Calomys laucha from 50 to 70 days old were

kept with males to reproduce. As no alterations in vaginal

swab were observed, after 5 days, males were separated

and females were divided into six groups that received,

by gavage, the following treatments: (i) 100 ll of water(Negative control Water); (ii) 100 ll of olive oil(vehicle control); (iii) 6.5 lg kg1 body weight (bw) ofdiethylstilbestrol (DES - positive control); (iv) 40 lgkg1 bw of BPA (BPA40); (v) 80 lg kg1 bw of BPA(BPA80); (vi) 200 lg kg1 bw of BPA (BPA200). Thevehicle for administration of DES and BPA was 100 ll of

olive oil. The treatment was given daily from the day

males were separated from females until parturition. After

this period, treatment ceased and breastfeeding occurred

normally.

The current oral reference dose established by the US

Environmental Protection Agency is 50 lg kg1 day1 forconsumption of BPA without deleterious effects

(Vandenberg et al., 2007). For this study, therefore, the

lowest dose of BPA was lower than this reference dose, and

the highest dose was based on the lowest dose used in

another study conducted by our group (300 lg kg1 datanot published). The oestrogenic potency of DES is 1001000 times higher than BPA (vom Saal & Welshons, 2006),

so a lower dose of this substance was used.

Pups were counted, weighed and sexed at birth. Their

weight was gauged every 10 days until postnatal day

(PND) 50. Anogenital distance was measured at birth and

at PND 21. Males and females were euthanised by cervical

dislocation on PND 70, after reaching full maturation of

the reproductive tract. The procedures used for

experimentation and euthanasia of the animals followed

the recommendations of Brazilian law #6638 of May 8,

1979, and also those of the US National Institute of

Health guide for the use of laboratory animals (1996).

Chemical substances were purchased from Sigma

(St. Louis, MO, USA).

Semen analyses

Spermatozoa were collected after approaching the repro-

ductive system by laparotomy. The cauda of both epi-

didymes and part of the vas deferens were removed,

ruptured with a 12 g 9 40 mm needle and placed in a

35 mm Petri dish containing 200 ll of M2 medium withHEPES (M7167) (Corcini et al., 2012) for sperm dilution.

Sperm quality evaluations were done as described below

after incubation of samples for 10 min at 37 C in M2medium.

For sperm motility evaluation, an aliquot (10 ll) con-taining spermatozoa was placed between a slide and a cover

glass, and warmed at 37 C for further observation under aphase contrast optical microscope (Olympus BX 41-PH-III

America INC, S~ao Paulo, Brazil). Sperm motility was

reported as the average of three evaluations carried out by

a trained technician using optical microscopy at 2009

(Tayama et al., 2006) and expressed as percentage (0100%). Sperm morphology was determined after counting

200 cells using phase contrast microscopy at 10009 (Tay-

ama et al., 2006). Normal spermatozoa were counted, as

well as defects of distal droplet, detached head and tucked

tail. The number of sperm recovered from the epididymis

was counted in a Neubauer chamber by calculating the

concentration of spermatozoa per ml of medium.

2 2013 Blackwell Verlag GmbHAndrologia 2013, xx, 18

Sperm damage in mice by in utero exposure to BPA J. Vilela et al.

Sperm DNA integrity, mitochondrial functionality,

DNA integrity, sperm membrane integrity and acrosome

integrity were held in an epifluorescence microscope

(Olympus BX 51; America INC, S~ao Paulo, Brazil), with

5 ll of solution with sperm under coverslip (18 918 mm), evaluating 200 cells per sample. Results were

expressed in percentage of functional cells over total of

evaluated cells. Sperm DNA integrity was evaluated with

acridine orange gauge (Evenson & Jost, 2000). Sperm

membrane integrity and acrosome integrity were evalu-

ated at 4009 with 450520 nm filtre wavelength. Spermmembrane integrity was evaluated using carboxyfluoresce-

in diacetate and propidium iodide (Harrison & Vickers,

1990) and in each slide, 200 cells were counted and classi-

fied as follows: intact (green fluorescence) or not intact

(either red or simultaneous red and green fluorescence).

Acrosome integrity was evaluated using FITC-PNA, after

counting 200 cells in dry slides. Acrosomes were classified

as follows: intact, when the sperm cell presented red fluo-

rescence, the acrosome presented green fluorescence and

the cells conformation was normal; or not intact, when

the sperm cell presented red fluorescence but the acro-

some was not evident or presented abnormal conforma-

tion (Jimenez et al., 2003). Results were expressed as

percentage of intact acrosome. Mitochondrial functional-

ity was evaluated by fluorescent rhodamine 123 (Johnson

et al., 1980).

For the in vitro penetration (IVP) test, fresh oocytes were

collected from the ovarium of prepubertal gilts obtained

from a local abattoir. Oocytes were collected and pro-

cessed; thirty oocytes were used for each sample and the

test followed the protocol described by Corcini et al.

(2012). Briefly, a M2 medium containing HEPES, 0.4%

bovine serum albumin (A3311) and approximately

2 9 106 spermatozoa per ml was used. Gametes were incu-

bated in a water bath (37 C) for 2 h. Following incuba-tion, oocytes were collected, washed, stained with Hoescht

33 342 (10 lg ml1) for 15 min at 38 C and thenobserved under an epifluorescence microscope (4009 mag-

nification). Penetration rate (%) was calculated based on

the relationship between the number of penetrated oocytes

and the total number of oocytes per sample and per

treatment. For each male, the number of penetrated

oocytes and of spermatozoa per oocyte was counted.

Statistics

Statistical analysis was carried out using Statistica 7.0

software. Descriptive statistics were calculated for all eval-

uated sperm variables. The continuous variables were

tested for normal distribution with ShapiroWilk anddata were compared by the KruskalWallis H-test (Krus-kalWallis one-way analysis of variance by ranks) withmultiple comparisons of mean ranks for all groups (Siegel

& Castellan, 1988).

Results

Of all sixty females, 41 sired litters. Mean birth weight

was 1.6 0.1 g and there was no statistical differencebetween the groups. One animal from the BPA40 group

was born dead and without three limbs (Fig. 1). Two ani-

mals from the BPA80 group presented male genitals and

female reproductive tract. No statistical differences were

found for number of males and females and proportion

of genders per treatment, which was 15.6 1.9 malesand 15.3 2.3 females (proportion 1:1). Number ofpups per litter also presented no statistical differences

between the groups, with a mean of 3.8 0.31 femalesand 2.2 0.48 males. No statistical differences werefound for anogenital distance (AGD), which was

3.48 0.06 mm.Spermatozoa were collected and analysed from a total

of 71 males, 16 being from water group, 8 from olive oil,

13 from DES, 15 from BPA40, 11 from BPA80 and 8

from BPA200. Number of spermatozoa increased signifi-

cantly in the olive oil group in relation to all other

groups (P < 0.05). Values tended to be lower in DES andBPA groups (Fig. 2) in relation to vehicle group, despite

being statistically equal to water group.

Sperm DNA integrity and acrosome integrity are pre-

sented in Table 1. No statistical differences were found

(a) (b)

Fig. 1 Newborn Calomys laucha from bisphe-

nol A (BPA) 40 group without three limbs.


J. Vilela et al. Sperm damage in mice by in utero exposure to BPA

for DNA integrity, but the values tended to drop in the

BPA200 group (P > 0.05). Acrosome integrity did notdiffer between vehicle group and treatment groups, but

values were significantly higher in the water group.

Normal sperm morphology was statistically reduced

only in the BPA200 group (P < 0.05). Although defectsof morphology did not present statistical differences

between groups, BPA200 showed higher values in distal

droplets and detached head, while DES and BPA200 pre-

sented more tucked tails than the other groups (Table 2).

Sperm mitochondrial integrity, motility and sperm

membrane integrity are presented in Table 3. Mitochon-

drial integrity was statistically equal in the water and

BPA40 groups and significantly lower (P < 0.05) in thevehicle, DES, BPA80 and BPA200 groups, which did not

differ statistically from each other. Sperm motility was

statistically reduced in BPA80 and BPA200 in relation to

the other groups and membrane integrity was signifi-

cantly reduced in BPA40 and BPA200 in relation to the

other groups (P < 0.05).

In vitro penetration rates were significantly reduced at

all BPA concentrations (Fig. 3). Number of penetrating

spermatozoa per oocyte was statistically lower only in the

BPA200 group (Fig. 4).

Discussion

Bisphenol A has shown conflicting responses in different

species and doses. It has been reported that litter size

remained consistent across BPA (50 and 1000 lg kg1)treatment groups and the vehicle (sesame oil) in mice,

but DES (5 lg kg1) decreased litter size both at birthand weaning (LaRocca et al., 2011). Studies have also

reported that in utero administration of doses of BPA

(0, 4 and 40 mg kg1 day1) through gavage had noeffects on growth and AGD of F1 rat offspring (Kobay-

ashi et al., 2002). Contrary to these observations, another

study reported that in utero administration of doses of

BPA (50 lg kg1 day1) through diet increased AGD inCD-1 mice offspring (Gupta, 2000). In this study, results

showed that in utero exposure to BPA and DES did not

affect litter size, weight of animals or the AGD. Discrep-

ancies between the results may be because of differences

Fig. 2 Number of spermatozoa for Calomys laucha exposed in utero

to water, olive oil, Diethylstilbestrol (DES) (6.5 lg kg1) and bisphenolA (BPA) (40, 80 and 200 lg kg1) (N = 71). Data expressed asMean SE. Values followed by different letters differ significantly byKruskalWallis test (P < 0.05).

Table 1 Sperm DNA integrity and acrosome integrity for Calomys

laucha exposed in utero to water, olive oil, Diethylstilbestrol (DES)

(6.5 lg kg1) and bisphenol A (BPA) (40, 80 and 200 lg kg1)(N = 71)

Treatment DNA Acrosome

Water 97.9 0.73a 89.6 2.24aOlive Oil 95.4 2.01a 50.0 2.38bDES 91.0 4.12a 43.6 4.08bBPA40 85.1 6.19a 48.1 3.29bBPA80 90.5 4.23a 48.7 2.52bBPA200 89.5 3.06a 49.2 2.71b

Results are expressed as Mean of percentage SE. Values followedby different letters in the same column differ significantly by Kruskal

Wallis test (P < 0.05).

Table 2 Sperm morphology for Calomys laucha exposed in utero to

water, olive oil, Diethylstilbestrol (DES) (6.5 lg kg1) and bisphenol A(BPA) (40, 80 and 200 lg kg1) (N = 71)

Treatment Normal

Distal

droplet

Detached

head

Tucked

Tail

Water 96.1 0.65a 1.1 0.38a 1.0 0.25a 2.0 0.59aOlive Oil 96.0 0.86a 1.8 0.89a 1.1 0.32a 0.9 0.49aDES 94.5 0.98a 1.3 0.42a 0.7 0.49a 4.0 0.96aBPA40 93.7 1.50a 1.5 0.61a 1.4 0.41a 3.4 1.64aBPA80 94.6 1.08a 1.1 0.30a 1.0 0.43a 3.9 1.12aBPA200 90.8 2.30b 3.8 1.01a 2.7 0.84a 4.4 2.93a

Data expressed as Mean of percentage SE. Values followed by dif-ferent letters in the same column differ significantly by KruskalWallis

test (P < 0.05).

Table 3 Sperm mitochondrial integrity (Mit). motility and membrane

integrity (Mem) for Calomys laucha exposed in utero to water, olive

oil, Diethylstilbestrol (DES) (6.5 lg kg1) and bisphenol A (BPA) (40,80 and 200 lg kg1) (N = 71)

Treatment Mit Motility Mem

Water 92.8 1.71a 87.5 1.71a 88.8 1.65aOlive Oil 35.2 6.24b 65.7 13.43a 77.2 3.63aDES 35.8 7.09b 78.8 2.27a 55.9 13.85aBPA40 61.8 9.20a 79.2 2.60a 65.5 5.09bBPA80 58.6 6.89b 61.8 7.11b 74.6 4.14aBPA200 56.1 7.20b 51.3 9.90b 70.6 4.15b

Data expressed as Mean of percentage SE. Values followed by dif-ferent letters differ significantly by Kruskal-Wallis test (P < 0.05).



among species and route and vehicle of administration of

the substances.

It is known that in utero exposure of DES in women is

a cause of several anatomical and functional disorders of

the genital tract (Laurent et al., 1998). In this study,

although neither BPA nor DES affected AGD, it was

observed that only 3 females that received DES by gavage

sired a litter, while this number was much higher in the

other groups. The dose of 6.5 lg kg1 of DES given tothe females may have caused anatomical, functional or

hormonal disorders. To verify this hypothesis, uteri of all

the female breeders and their litters were collected for

future histological analyses.

Studies indicate that BPA exposure results in perma-

nent alterations in androgen-target tissues (Maffini et al.,

2006). Corroborating this hypothesis, an increase in pros-

tate duct volume (Timms et al., 2005), enlarged prostates

and decreased epididymal weight (Gupta, 2000) have

been previously reported in mice. In this study,

80 lg kg1 of BPA induced malformation of the repro-ductive system; additionally, 40 lg kg1 of BPA induced

limb malformation in one animal. Other studies have

reported teratogenic effects of BPA in Xenopus laevis

(Iwamuro et al., 2003) and early mice embryos in vitro

(Pei et al., 2003), but this study demonstrates that doses

lower than that accepted by the US Environmental

Protection Agency may cause death and malformation in

foetuses of rodents.

Sperm DNA integrity has been shown to reduce in a

doseresponse manner with urinary BPA in men (Meekeret al., 2010). It has also been reported that in utero expo-

sure to endocrine disruptors influences the embryonic

testis and causes epigenetic effects such as DNA methyla-

tion, resulting in abnormal germ-cell differentiation that

influences adult spermatogenic capacity and male fertility

in rats (Lenzi et al., 1996). The doses used in this study

did not affect sperm DNA, but BPA tended to reduce

DNA integrity, so it is possible that higher doses may

cause a significant decrease in this parameter.

The effect of olive oil on acrosome integrity, mitochon-

drial integrity and increasing number of sperm lead us to

question whether olive oil was the best vehicle for admin-

istration of BPA. Many genes involved in fatty acid

metabolism are regulated by a family of three members of

the nuclear peroxisome proliferator-activated receptors

(PPARs), which regulate glucose homeostasis, lipid

metabolism and inflammation. There is indication that

oleic, linoleic and palmitic acids, which are present in

the olive oil, are ligands of PPARs (revised by Kaput

& Rodriguez, 2004). Since most studies do not compare

vehicle treatment with negative control (water),

comparing only treatment with BPA and other oestrogenic

compounds and the vehicle used (Nagao et al., 1999;

Kobayashi et al., 2002; Toyama & Yuasa, 2004; Salian

et al., 2009; LaRocca et al., 2011), our study may provide

new information about the use of oily vehicles for

administration of BPA, which may in fact protect the

cells against the epigenetic effect of BPA. Moreover, in

spite of the improving effects of olive oil, BPA still caused

a reduction in sperm parameters, indicating its inhibiting

effect. Sperm morphological abnormalities have been

reported in rats prenatally exposed to BPA, which

presented cytoplasmic droplets in the mid-piece, principal

and head regions of the spermatozoa (Salian et al., 2009).

In this study, droplets were found in the distal region of

the cell. However, these abnormalities may not impair

C. laucha reproduction, since normal sperm morphology

of this species has been reported by Corcini et al. (2012)

to be equal to 90.2 6.6%, which is lower than theBPA200 group in this study. Sperm motility has also been

shown to decrease in rats perinatally exposed to BPA

(Salian et al., 2009), and lower motility related to urinary

BPA in a doseresponse manner in men has beenreported (Li et al., 2011). Corroborating these findings, in

Fig. 3 In vitro penetration (IVP) rates for Calomys laucha exposed in

utero to water, olive oil, Diethylstilbestrol (DES) (6.5 lg kg1) andbisphenol A (BPA) (40, 80 and 200 lg kg1) (N = 71). Data expressedas Mean of percentage SE. Values followed by different lettersdiffer significantly by KruskalWallis test (P < 0.05).

Fig. 4 Number of penetrating spermatozoa per oocyte for Calomys

laucha exposed in utero to water, olive oil, Diethylstilbestrol (DES)

(6.5 lg kg1) and bisphenol A (BPA) (40, 80 and 200 lg kg1)(N = 71). Data expressed as Mean SE. Values followed by differentletters differ significantly by KruskalWallis test (P < 0.05).



this study, sperm motility was significantly decreased in

groups BPA80 and BPA200 in relation to water and olive

oil.

The effect of BPA on plasma membrane integrity is

demonstrated for the first time in this study. Our results

for the water group were similar to those found by Cor-

cini et al. (2012), who studied in vitro assays for vesper

mice sperm and reported normal membrane integrity as

88.7 9.6% for C. laucha.All the parameters discussed above may influence the

IVP test. The IVP test presents high specificity, detecting

sub-fertile males unable to sire a litter after natural mat-

ing by detecting those failing to penetrate swine oocytes

in vitro (Corcini et al., 2012). Since C. laucha sperm head

morphology is similar to that of other mammals (Lasserre

et al., 2000), it has been shown that porcine oocytes are

useful heterologous substrates for in vitro fertilisation

studies of this species (Lasserre et al., 2000; Corcini et al.,

2012). This is the first study of IVP rates related to BPA.

In this study, BPA significantly reduced penetration rates

at all doses given. However, the reason for this reduction

in IVP rates could not be determined. As heterologous

oocytes were used, it is possible that BPA affected an

important factor for the spermatozoa to penetrate the

oocyte that was not analysed in this study. Corcini et al.

(2012) found IVP rates for C. laucha equal to 39.8%,

which is lower than our control groups, but higher than

treatment groups. However, to confirm the reduction of

fertility by BPA and DES, natural mating studies need to

be performed.

The mechanisms of action of BPA are not fully under-

stood. The adverse effects of BPA on sperm parameters

may be due to a direct effect on the testes, acting as an

androgen receptor (AR) antagonist that interrupts normal

AR binding activity (Wetherill et al., 2007). Also, BPA

may alter spermatogenesis by disrupting the hypothala-

muspituitarytestes axis, altering the function of Leydigcells and reducing testosterone biosynthesis (Takao et al.,

2003).

This study shows that prenatal exposure to BPA causes

permanent alterations in semen quality of C. laucha,

while other studies show that effects of adult and perina-

tal administration of BPA are transitory (Nagao et al.,

1999; Kuwada et al., 2002; Toyama & Yuasa, 2004). The

differences between in utero and perinatal administration

of BPA may occur because in utero exposure to BPA

affects the developing testes, thus affecting germ cells,

while perinatal exposure affects the already formed organ.

Another important observation in this study is that

most of the parameters were affected only by the highest

dose of BPA. Thus, data suggest C. laucha is more resis-

tant to low doses of BPA than other rodents; this may be

because wild species present higher genetic variability,

making them more resistant to a series of infectious and

stressful agents.

In conclusion, it is shown for the first time in vivo that

low doses of BPA may cause malformation of foetuses.

In utero exposure to high doses of BPA permanently

reduced normal sperm morphology, sperm membrane

integrity, sperm motility and IVP rates of adult C. laucha

males. This species may act as a bioindicator of BPA pol-

lution in natural environments. Natural mating experi-

ments must be conducted to confirm whether these

alterations reduce the fertility of the animals.

Acknowledgements

We thank the ReproPel team from the University of Pelo-

tas for help with the semen analyses and CAPES (Coorde-

nac~ao de Aperfeicoamento de Pessoal de Nvel Superior,Braslia, DF, Brazil) for financial support.

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Sperm impairments in adult vesper mice (Calomys laucha) caused by in utero exposure to bisphenol A

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