I f A J E S a b c d a A R R A A K F F P F M 1 o [ a p p g r A g f m R o ( h 0 Reproductive Toxicology 62 (2016) 1–8 Contents lists available at ScienceDirect Reproductive Toxicology j ourna l h o mepa ge: www.elsev ier .com/ locate / reprotox n utero and lactational exposure to fluoxetine delays puberty onset in emale rats offspring lice Hartmann dos Santosa, Milene Leivas Vieiraa, Nathália de Azevedo Camina, anete Aparecida Anselmo-Francib, Graziela Scalianti Ceravoloa, Gislaine Garcia Pelosi a, stefânia Gastaldello Moreiraa, Ana Carolina Inhasz Kissc, uzana de Fátima Paccola Mesquitad, Daniela Cristina Ceccatto Gerardina,∗ Department of Physiological Sciences, State University of Londrina, 86051-980 Londrina, Paraná, Brazil Department of Morphology, Estomatology and Physiology, University of São Paulo, Ribeirão Preto, São Paulo, Brazil Department of Physiology, São Paulo State University, Botucatu, São Paulo, Brazil Department of Biology, State University of Londrina, 86051-980 Londrina, Paraná, Brazil r t i c l e i n f o rticle history: eceived 11 May 2015 eceived in revised form 11 March 2016 ccepted 7 April 2016 vailable online 14 April 2016 eywords: a b s t r a c t Depression is one of the most prevalent disorders in the world and may occur during pregnancy and postpartum periods. Fluoxetine (FLX) has been widely prescribed for use during depression in pregnancy and lactation. This study aimed to investigate if in utero and lactational exposure to FLX could compro- mise reproductive parameters in female offspring. Wistar rats received, by daily gavage, FLX 5 mg/kg or 0.3 ml of water (control group) from the first gestational day until weaning (21 days). Assessments in the female offspring included: body weight, anogenital distance, vaginal opening, first estrus, estrous cycle, emale ertility uberty luoxetine aternal behavior reproductive organs weight, uterine morphometric analyses, ovarian follicle and corpora lutea counting, estradiol plasmatic concentration, sexual behavior, maternal behavior and fertility test. Exposure to FLX delayed the puberty onset in female pups. The present study demonstrated that developmental exposure to FLX can deregulate the neuroendocrine hormonal control of female offspring during prepubertal and pubertal periods. © 2016 Elsevier Inc. All rights reserved. . Introduction Pregnancy and postpartum period are risk factors for the devel- pment or exacerbation of mental disorders, such as depression 1], due to the emergence of biological, physiological and social lterations. Approximately 40% of women that suffer from post- artum depression develop the symptoms during gestation [2]. The revalence of perinatal depression is 10–20% [1,3]. Abbreviations: 5-HT, serotonin; DA, dopamine; NA, noradrenaline; GABA, amma-aminobutyric acid; CTR, control; FLX, fluoxetine; SSRIs, selective serotonin euptake inhibitors; GD, gestational day; LD, lactational day; PND, postnatal day; GD, anogenital distance; VO, vaginal opening; HPG axis, hypothalamic-pituitary- onadal axis; GnRH, gonadotropin release hormone; LH, luteinizing hormone; FSH, ollicle stimulating hormone; LM, lordosis magnitude; LQ, lordosis quotient; MB, aternal behavior; ANCOVA, analysis of covariance; ANOVA, analysis of variance; MANOVA, repeated measures analysis of variance. ∗ Corresponding author at: Department of Physiological Sciences, State University f Londrina-UEL, 86051-980 Londrina, Paraná, Brazil. E-mail addresses: dcgerardin@uel.br, danigerardin@yahoo.com.br D.C.C. Gerardin). ttp://dx.doi.org/10.1016/j.reprotox.2016.04.006 890-6238/© 2016 Elsevier Inc. All rights reserved. Fluoxetine (FLX) is a selective serotonin reuptake inhibitor (SSRI) antidepressant and is the drug of choice during pregnancy due to its relative selectivity of action, efficacy, reduced side effects [4] and absence of morphological teratogenic activity [5]. FLX and its active metabolite, norfluoxetine, readily crosses the placenta in humans [6] and in experimental animals [7] and are also excreted in human milk [6]; therefore fetuses and neonates are exposed to these substances during important stages of development. Fetal exposure to SSRI has been associated to low uterine fetal growth and symptoms related to changes in motor and somatosen- sory systems development [8]. Serotonin (5-HT) is known to be a trophic agent for the migration and synaptogenesis of monoamin- ergic neurons during brain development [9], whereas in adult life it is an important factor for neurogenesis and neuronal maintenance in the central nervous system [10]. Thus, it can be suggested that exposure to FLX during neurodevelopment may influence not only the 5-HT system, but all the monoaminergic systems. As a matter of fact, it has been reported that perinatal exposure to FLX can impair the dopaminergic (DA) system [11]. dx.doi.org/10.1016/j.reprotox.2016.04.006 http://www.sciencedirect.com/science/journal/08906238 http://www.elsevier.com/locate/reprotox http://crossmark.crossref.org/dialog/?doi=10.1016/j.reprotox.2016.04.006&domain=pdf mailto:dcgerardin@uel.br mailto:danigerardin@yahoo.com.br dx.doi.org/10.1016/j.reprotox.2016.04.006 2 ductiv t [ a r c p p r s c i p l b t h b i i t t u r 2 2 m a e d ( z e g S b a U m i l u A n r b c s d - - a w A.H. dos Santos et al. / Repro Additionally, 5-HT system may influence the reproductive func- ion of vertebrate due to its communication with sex steroid system 12]. This interaction between the systems occurs through estrogen nd progesterone receptors located in serotoninergic central neu- ons, demonstrating an important signaling pathway. These 5-HT entral neurons can modulate several neural processes, including ituitary secretion and sexual behavior. In this way, estrogen and rogesterone can interfere with these functions since these neu- ons are sensitive to ovarian hormones. Thus, the serotoninergic ystem may integrate signals from the hormonal system with the entral nervous system. Similarly to 5-HT, DA also regulates reproductive behaviors nteracting with steroid hormones. Interactions between estradiol, rogesterone and DA in the ventromedial nucleus of the hypotha- amus regulate the lordosis reflex in rats [13]. Regarding maternal ehavior (MB), the main components of the neural circuitry include he pre-optic area, the nucleus accumbens and other limbic and ypothalamic structures [14]. Despite the predominant role played y DA on MB [15], there are evidences that 5-HT is also involved n this behavior [16]. However, there are still few works evaluat- ng the impact that developmental exposure to SSRI could have on he reproductive and central nervous systems functions. Based on hese considerations, this study was carried out in order to eval- ate if in utero and lactational exposure to FLX could disrupt the eproductive development of female offspring. . Material and methods .1. Animals and treatment This study is part of a main study conducted in the Depart- ent of Physiological Sciences, State University of Londrina, which imed to investigate reproductive, cardiovascular and neurological ndpoints after developmental exposure to FLX. The experimental esign adopted followed principles described in the guideline 426 Developmental Neurotoxicity Studies) published by the Organi- ation for Economic Co-Operation and Development [19] which, xcept for the lack of pre-mating treatment, complies with the uideline 443 (Extended One-Generation Reproductive Toxicity tudy) from the same agency. All animal procedures were approved y the UEL Ethics Committee for Animal Research (16166.2012.12). The experimental design is depicted in Fig. 1. A total of 17 male nd 35 female Wistar rats (85-90 days) from the colony of the State niversity of Londrina (UEL) were used as parental generation. Ani- als were group housed five per cage in a polypropylene cage kept n a controlled environment with temperature at 21 ± 2 ◦C; 12 h ight/dark cycle (lights on at 6:00 a.m.) and had free access to reg- lar lab chow (NuvitalTM, Paraná, Brazil) and tap water bottles. utoclaved wood shaver bedding was used to prevent contami- ation from mycoestrogens in corncob bedding. Female and male ats were maintained separated by sex during 15 days prior to the eginning of mating. Rats were mated (2 females and 1 male per age) and gestational day (GD) 0 was determined if there were perm and estrus phase cells in vaginal smears. Dams were ran- omly divided into 2 groups: Control (CTR): 17 dams received tap water daily, by gavage, from GD 0 to postnatal day (PND) 21; FLX: 18 dams received FLX (5 mg/kg, DaforinTM oral solution, EMS Laboratory, Brazil), by gavage, from GD 0 to PND 21. Dams were daily treated at 11:00-13:00 p.m. and doses were djusted each 3 days according to weight. In humans, FLX prescription ranges from 20 to 80 mg/day, which ould correspond to approximately 0.29–1.14 mg/kg. Considering e Toxicology 62 (2016) 1–8 that the precautionary principle considers animals more resistant than humans, higher doses are tested in animals. Preliminary stud- ies of our group showed that the dose of 10 mg/kg decreased the number of pups per litter (data not shown). Vorhees et al. reported that prenatal exposure to 12 mg/kg FLX resulted in a significant increase in offspring mortality from birth to PND 7 with no alter- ations observed in pups exposed to 5 mg/kg of FLX [17]. Bairy et al. demonstrated that prenatal exposure to 12 mg/kg FLX resulted in a significant reduction in birth weight and that this alteration was not observed at 8 mg/kg dose [18]. In this way, the dose of 5 mg/kg was chosen to ensure no influence on litter size and weight. At birth (PND 0), pups were counted, the sex determined and the litters were weighed. On PND 4, litters were culled to 10 pups keeping 5 males and 5 females whenever possible. From each litter, 1–2 female pups were used for this study. The anogenital distance, body weight and vaginal opening were observed in the 2 female pups per litter, whenever possible. After that, one female was allo- cated to the behavioral evaluation and the other to the assessment of non-behavioral reproductive parameters. 2.2. Parameters analyzed in female offspring during development (PND 0–60) 2.2.1. Body weight Female pups body weight was measured on PND 0, 7, 14 and 21 (CTR: 17 litters and FLX: 18 litters). This data is expressed as litter mean. 2.2.2. Physical sexual development The anogenital distance (AGD, distance from the anus to the genital tubercle) was measured on PND 0 and 21 (CTR: 17 litters and FLX: 18 litters). AGD was normalized through its division by the cube root of body weight. From PND 30 on, females from both groups were daily evaluated for vaginal opening (VO, CTR: 27 litters and FLX: 28 litters) in order to determine the day in which complete VO occurred. In this day, females were weighed. Starting from the day of VO, daily vaginal smears were collected to detect the day of the first estrus (CTR: 11 litters and FLX: 11 litters), characterized by the predominance of cornified epithelial cells [20]. These parame- ters were evaluated in 2 littermates and data are expressed as litter means. 2.3. Parameters analyzed in female offspring during adulthood 2.3.1. Estrous cycle evaluation On PND 75, the estrous cyclicity of female rats (CTR: 12 rats and FLX: 11 rats) was assessed through vaginal smears collected over a period of 15 days as previously described in Ref. [21]. The cycle phases were cytologically determined by the follow- ing characteristics: predominance of nucleated epithelial cells (proestrus); predominance of cornified epithelial cells (estrus); presence of cornified and nucleated epithelial cells and leukocytes (metaestrus); predominance of leukocytes (diestrus). The total fre- quency of each phase was used to calculate the total length (in days) of the proestrus, estrus, metaestrus and diestrus and the estrous cycle length. 2.3.2. Collection of tissues and organs After the estrous cycle evaluation and during an estrus phase (PND 90–95), females (n = 10/group) were weighed, deeply anes- thetized with thiopental and laparatomized. Blood samples were collected from abdominal aorta for quantification of plasmatic estradiol. The ovaries and uteri (with fluid) were collected and weighed. After that, they were euthanized by decapitation. The right ovary and the medial portion of the right uterine horn were fixed in Bouin, dehydrated in ethanol and embedded in Paraplast® A.H. dos Santos et al. / Reproductive Toxicology 62 (2016) 1–8 3 Fig. 1. Diagram of experimental design. PND: postnatal day; AGD: anogenital distance; LD: lactational day. F s ± S.E l ( o a b g ( d ( ( O s 2 s s p e m 1 S a r ig. 2. Body weight (A) and anogenital distance (B) of female pups. Values are mean itters. Sigma, St. Louis, MO, USA). Six sections (5 �m) per animal were btained, mounted on glass slides and stained with hematoxylin nd eosin (CTR n = 6/FLX n = 8). There was an interval of 100 �m etween the first 3 and the last 3 sections. In ovary, the corpora lutea and ovarian follicles (primordial, rowing, antral and atretic) were counted under light microscopy OSM-223287, Olympus; 100 × and 400 × magnifications) in accor- ance to Pedersen and Peters [22], as described in Ref. [23]. In uterine horn, it was measured the luminal epithelial height 400× magnification) and thickness of the endometrial stroma 100× magnification). For this, a light microscope (OSM-223287, lympus) coupled to a digital camera and a computer with Bel view oftware was used. .3.3. Plasmatic estradiol quantification Blood samples were collected from abdominal aorta into yringes containing heparin. Immediately after collection, blood amples were centrifuged (2500 rpm for 20 min at 2 ◦C) and the lasma was frozen until assayed for determination of serum stradiol levels. The estradiol plasmatic concentration (10 ani- als/group) was measured by radioimmunoassay, using the 7�-Estradiol (E2) Double Antibody RIA kitTM (MP Biomedicals, anta Ana, CA, USA). The limit of detection was 1.2 pg/ml and intra- ssay and inter-assay coefficients of variation were 2.5% and 7.0%, espectively. .M. RMANOVA (p > 0.05). CTR = saline, n = 17 litters; FLX = Fluoxetine 5 mg/kg, n = 18 2.3.4. Sexual behavior evaluation Sexual behavior (CTR: 17 females and FLX: 16 females) was ana- lyzed during the dark phase of a reversed light/dark cycle, under dim red light. The animals were allowed a 15-days period of adap- tation to the reversed light/dark cycle before the evaluation. The sexual behavior was assessed in cycling rats 3–4 h after the diagno- sis of proestrous through vaginal smear. The analysis always started 4 h after the onset of darkness and the behavior was recorded by a video camera linked to a monitor in an adjacent room. Sexually experienced males from the State University of Londrina colony (i.e., not the siblings obtained in our department) were used in these tests, which lasted until the occurrence of ten mounts. Results were expressed as the lordosis quotient (LQ, number of lordosis/ten mounts × 100) as well as the frequency of each lordosis magnitude (LM, on a scale of 0–3). The classification was as follows: 0 absence of lordosis; 1 the female showed little flex of spine, head and hips slightly elevated from floor; 2 the female showed spinal flex and head raise close of an angle of 30◦ with the floor; 3 maximum lor- dosis, with accented spinal flex and the head inclined at an angle of 45◦ or more relative to the floor. 2.3.5. Maternal behavior To investigate the effects of in utero and lactational exposure to FLX on mother-pup interaction, females were mated for analyzing the maternal behavior. Two types of MB analyses were conducted in 4 A.H. dos Santos et al. / Reproductive Toxicology 62 (2016) 1–8 F le pup m trus - i d 2 d w r f p a w i r m s 2 a n B t g u p ig. 3. Percentual representation of vaginal opening (A) and first estrus (B) of fema eans ± S.E.M. ANOVA (*p < 0.05). Vaginal Opening - CTR n = 27; FLX n = 28; First Es ndependent groups of dams and both were recorded at lactational ay (LD) 5 between 7:30 a.m. and 1:30 p.m. .3.5.1. Maternal behavior evaluation after pup removal. On the test ay, all pups were removed from their home cage and the nest as destroyed (CTR n = 10/FLX n = 9). After 30 min, the pups were eturned to the cage and mother-pup interaction was recorded or 30 min. Latency for retrieval behavior and total time grouping, up grooming, self-grooming, crouching, off pups (defined as the mount of time that the rat spent without any kind of interaction ith pups regardless of her position in the cage), and nest build- ng were quantified. Full maternal behavior was scored if dams etrieved all pups to the nest and nursed them for 3 consecutive inutes. All behavioral analyses were performed using Etholog oftware [24]. .3.5.2. Undisturbed mother-pup interaction evaluation. Mother nd litter interaction was recorded on their home cage for 6 h (CTR = 11/FLX n = 10). Videotaping began at 7:30 a.m. in the light phase. ehavior was scored every minute during this period according to he following categories: nursing, off pups, retrieval behavior, pup rooming and self-grooming. Total number of observations was sed to calculate the percentage of observations in self-grooming, up-grooming, nursing, building nest, retrieval and off pups. s. Average day of vaginal opening (C) and first estrus (D) of female pups. Values are CTR n = 11; FLX n = 11. PND = postnatal day; CTR = saline; FLX = Fluoxetine 5 mg/kg. 2.3.6. Fertility test On LD 5, after the MB analyses, female rats were deeply anes- thetized with thiopental (CTR n = 17/FLX n = 16). After that the uterus and ovaries were collected, they were euthanized by decapi- tation and the numbers of corpora lutea (by gross morphology) and implantation sites were counted. From these results, the following parameters were calculated: - implantation rate: number of implantation sites/number of cor- pora lutea × 100; - pre-implantation loss rate: number of corpora lutea—number of implantation sites/number of corpora lutea × 100; - post-implantation loss rate: number of implantation sites—number of live fetuses/number of implantation sites × 100; - fetal viability: number of live fetuses/number of implantation sites × 100. 2.4. Statistical analysis Initially, an exploratory analysis was conducted to evaluate normal distribution (Shapiro-Wilk test) and homogeneity of vari- ance (Levene’s test) of each variable. Variables that presented normal distribution and homogeneity of variance were analyzed by ANOVA. In the absence of normal distribution and/or homo- geneity of variance, variables were transformed in order to achieve the criteria for parametric analysis. Conversely, for the other vari- ables Mann-Whitney test was performed. For pups’ body weight A.H. dos Santos et al. / Reproductive Toxicology 62 (2016) 1–8 5 Table 1 Estrous cyclicity evaluation in the female offspring exposed to fluoxetine during gestational and lactational periods. Estrous cycling (days) CTR [n = 12] FLX [n = 11] Estrous cycle length 3.75 (3.75–5.00) 3.75 (3.56–5.00) Frequency of proestrus 4.00 (4.00–5.00) 4.00 (3.00–4.00) Frequency of estrus 4.00 (3.00–4.00) 4.00 (3.00–4.25) Frequency of metestrus 3.00 (2.00–3.00) 3.00 (3.00–3.00) Frequency of diestrus 4.00 (4.00–5.00) 4.00 (4.00–5.00) V t s ( ( t ( b l p 3 3 3 f 3 ( 3 w [ [ p t 3 d [ n g p 3 ( 3 c d [ 1 1 i 3 a e r c Table 2 Body weight and wet organs weight from female rats at PND 90–95. Weight (g) CTR [n = 12] FLX [n = 11] Body 226.89 ± 7.92 217.35 ± 5.92 Uterus 0.48 ± 0.06 0.56 ± 0.07 Right ovary 0.05 ± 0.003 0.06 ± 0.003 Left ovary 0.05 ± 0.003 0.05 ± 0.003 Values are expressed as means ± S.E.M. Numbers in brackets represent the number of animals/group. CTR = saline, FLX = Fluoxetine 5 mg/kg. Body weight was used as a co-variate in the analysis of organs weight (ANCOVA, p > 0.05). Table 3 Ovarian follicle and corpora lutea counting and uterine morphometric analyses of female rats in PND 90–95. Parameters (count) CTR [n = 6] FLX [n = 8] Primordial follicles 5.19 ± 0.41 4.35 ± 0.54 Growing follicles 4.36 ± 0.40 4.06 ± 0.54 Antral follicles 4.61 ± 0.38 3.63 ± 0.60 Atretic follicles 3.53 ± 0.42 3.56 ± 0.42 Corpora lutea 11.39 ± 0.45 11.85 ± 0.35 Parameters (�m) CTR [n = 6] FLX [n = 8] Thickness of uterine epithelium 24.03 ± 2.49 24.86 ± 2.24 Thickness of endometrial stroma 913.39 ± 52.10 926.94 ± 83.13 alues are expressed as median (1st–3st quartile). Numbers in brackets represent he number of animals/group. CTR = saline, FLX = Fluoxetine 5 mg/kg. There were no ignificant differences between groups, Mann-Whitney (p > 0.05). PND 0, 7, 14, 21) and AGD (PND 0, 21), repeated measures ANOVA RMANOVA) was applied with day as the within-subject factor and reatment as the between-subjects factor. For wet organs weight PND 90–100), analysis of covariance (ANCOVA) was applied with ody weight as covariate. The lordosis reflex magnitude was ana- yzed by Fisher’s test. Differences were considered significant if < 0.05. . Results .1. Parameters analyzed in dams .1.1. Body weight Maternal body weight during gestation and lactation was unaf- ected by FLX treatment (data not shown, RMANOVA, p > 0.05). .2. Parameters analyzed in female offspring during development PND 0–60) .2.1. Body weight and physical development Body weight of female pups during the first 3 weeks of age as not influenced by FLX exposure as indicated by RMANOVA F(2.01,66.32) = 2043; p = 0.138]. There was only an effect of age F(2.01,66.32) = 1970.373; p = 0.0001] reflecting the weight gain of ups (Fig. 2A). Age was also the only significant factor observed in he AGD [F(1,33) = 1248.423; p = 0.0001] (Fig. 2B). .2.2. Physical sexual development The repeated exposure to FLX cause significant alteration in ays of VO (CTR: 33.78 ± 0.36, n = 27; FLX: 35.83 ± 0.49, n = 28) F(1,53) = 11.374; p = 0.001] and first estrus (CTR: 33.71 ± 0.44, = 11; FLX: 35.68 ± 0.73, n = 11) [F(1,20) = 5.361; p = 0.031] are iven in Fig. 3. The in utero and lactational exposure to FLX delayed uberty onset in female offspring. .3. Parameters analyzed in female offspring during adulthood PND 75-LD 5) .3.1. Estrous cycle and estradiol plasmatic concentration FLX exposure did not affect the regular pattern of estrous yclicity (Table 1) neither the plasmatic concentration of estra- iol (pg/ml; CTR: 180.08 ± 36.31, n = 10; FLX: 267.53 ± 50.33, n = 10) F(1,18) = 1.985; p = 0.176], FSH (ng/ml; CTR: 1.10 ± 0.25, n = 9; FLX: .03 ± 0.13, n = 9) [F(1,16) = 0.71; p = 0.793] and LH (ng/ml; CTR: .44 ± 0.97, n = 9; FLX: 0.38 ± 0.05, n = 9) [F(1,16) = 1.18; p = 0.293] n adulthood on estrus phases. .3.2. Reproductive organs weight and histology The final body weight and reproductive organs weight of dult female are presented in Table 2. No significant differ- nce as a result of in utero and lactational FLX exposure were evealed in body weight [F(1,21) = 0.904, p = 0.353]. ANCOVA indi- ated that the wet weight of uterus [F(1,21) = 1.599, p = 0.221] and Values are expressed as means ± S.E.M. Numbers in brackets represent the number of animals/group. CTR = saline, FLX = Fluoxetine 5 mg/kg. There were no significant differences between groups, ANOVA (p> 0.05). right [F(1,21) = 0.962; p = 0.338) and left [F(1,21) = 0.955; p = 0.356] ovaries were similar between groups. Counting of ovarian follicle and corpora lutea as well as uterine morphometric analyses are given in Table 3. ANOVA indicated lack of treatment effect for all these evaluated parameters from FLX group when compared to CTR (p < 0.05). 3.3.3. Sexual behavior The sexual behavior results are shown in Table 4. In utero and lactational exposure to FLX did not influence the parameters from this evaluation. 3.3.4. Maternal behavior 3.3.4.1. Maternal behavior evaluation after pup removal. ANOVA indicated that FLX exposure did not affect the parameters evalu- ated, i.e. retrieval behavior (total pups: [F(1,14) = 1.671; p = 0.217]), total time grouping [F(1,15) = 1.786; p = 0.201], pup groom- ing [F(1,15) = 0.333; p = 0.573], self-grooming [F(1,10) = 0.595; p = 0.458], crouching [F(1,15) = 0.075, p = 0.986], off pups [F(1,15) = 0.098; p = 0.949] and nest building [F(1,14) = 0.167; p = 0.689] (Table 5). 3.3.4.2. Undisturbed mother-pup interaction evaluation. ANOVA indicated lack of FLX exposure effect for all the parame- ters evaluated, i.e. nursing [F(1,16) = 0.061; p = 0.940], off pups [F(1,16) = 0.082; p = 0.930], retrieval [F(1,16) = 3.452; p = 0.083], pup grooming [F(1,16) = 0.974; p = 0.338], self-grooming [F(1,16) = 0.153; p = 0.700] and nest building [F(1,16) = 1.308; p = 0.270] (CTR group: n = 10; FLX group: n = 9, p > 0.05; Table 5). 3.3.5. Fertility test The fertility test parameters are given in Table 6. They were not influenced by FLX exposure as indicated by Mann-Whitney test p > 0.05. 4. Discussion The present study evaluated the effects of in utero and lacta- tional exposure to FLX on physical and reproductive parameters in 6 A.H. dos Santos et al. / Reproductive Toxicology 62 (2016) 1–8 Table 4 Sexual behavior of female rats at PND 90–95. Parameters CTR [n = 17] FLX [n = 16] Lordosis quocient 100.00 ± 0.00 99.38 ± 0.63 Lordosis reflex magnitude (LRM) (% of observations) 0 0.00% (0/170) 0.63% (1/160) 1 1.76% (3/170) 4.38% (7/160) 2 34.71% (59/170) 26.88% (43/160) 3 63.53% (108/170) 68.13% (108/160) Lordosis quocient values are expressed as means ± S.E.M. and were analyzed by ANOVA. LRM values are expressed as percentage of observations and were analyzed by Fisher’s test. Numbers in brackets represent the number of animals/group. Numbers in parenthesis represent the number of observations presented in each lordosis grade/total of observations. CTR = saline, FLX = Fluoxetine 5 mg/kg. There were no significant differences between groups p > 0.05. Table 5 Maternal behavior observations of rats from CTR and FLX groups. MB after pup removal CTR [n = 10] FLX [n = 9] Time to retrieve the first pup (s) 49.11 ± 13.07 (10/10) 78.33 ± 25.34 (8/9) Time to retrieve half of the litter (s) 175.49 ± 30.00 (10/10) 139.06 ± 31.64 (8/9) Time to retrieve all pups (s) 352.00 ± 66.98 (8/10) 246.84 ± 46.17 (8/9) Total time grouping (s) 163.76 ± 19.26 (10/10) 213.56 ± 33.15 (8/9) Total time self grooming (s) 19.59 ± 4.08 (8/10) 14.54 ± 4.19 (5/9) Total time pup-grooming (s) 490.15 ± 70.62 (10/10) 546.35 ± 66.03 (9/9) Total time crouching (s) 372.98 ± 125.61 (6/10) 369.06 ± 169.98 (3/9) Total time off pups (s) 729.48 ± 86.17 (10/10) 737.55 ± 88.88 (9/9) Total time nest building (s) 147.36 ± 34.80 (7/10) 168.67 ± 38.72 (7/9) Undisturbed MB CTR [n = 11] FLX [n = 10] Off pups (% of observations) 14.75 ± 1.02 (11/11) 14.97 ± 2.15 (10/10) Grouping (% of observations) 1.91 ± 0.33 (11/11) 2.25 ± 0.27 (10/10) Total nursing (% of observations) 69.75 ± 1.19 (11/11) 69.51 ± 3.51 (10/10) Self-grooming (% of observations) 2.04 ± 0.25 (11/11) 2.19 ± 0.31(10/10) Pup-grooming (% of observations) 11.51 ± 1.02 (11/11) 10.06 ± 1.06 (10/10) Nest building (% of observations) 0.86 ± 0.14 (11/11) 1.17 ± 0.23 (9/10) Values are expressed as means ± S.E.M. with the number of animals that displayed the behavior per total number of animals in the group given in parenthesis. Numbers in brackets represent the number of animals/group. The parameters were calculated using only animals that presented that behavior. CTR = saline, FLX = Fluoxetine 5 mg/kg. There were no significant differences between groups (ANOVA, p > 0.05). Table 6 Fertility parameters evaluated in adult female rats from CTR and FLX groups on lactational day 5. Parameters CTR [n = 17] FLX [n = 16] Implantation rate 61.36 (52.59–66.30) 60.87 (54.55–76.19) Pre-implantational loss (%) 38.64 (33.70–47.41) 39.13 (23.81–45.45) Post-implantational loss (%) 4.17 (0.00–19.55) 0.00 (0.00–7.14) Fetal viability rate 95.38 (80.45–100.00) 100.00 (92.86–100.00) V t the s t t l e w m m s o s 7 a i i a s p c alues are expressed as median (1st–3st quartile). Numbers in brackets represen ignificant differences between groups (Mann-Whitney, p > 0.05). he female offspring. Firstly, clinical signs and non-invasive indica- ors of maternal toxicity (such as changes in behavior—agitation, ethargy and hyperactivity; autonomic signs—lacrimation, pilo- rection, pupil-size, unusual respiratory patterns; body weight) ere evaluated during the FLX treatment period and no signs of aternal general toxicity were observed. The best indicator for the evaluation of pups’ physical develop- ent has been suggested to be the body weight gain. In the present tudy, FLX exposure did not induce body weight changes in female ffspring. Our results are in agreement to those observed by several tudies with different FLX employed doses, such as 1 [17], 5 [17], .5 [25] and 16 mg/kg [26]. However, it was observed, at doses of 10 nd 12 mg/kg, an increase [27] and a decrease [17,18] respectively n the body weight. These divergences can be related to differences n the exposure protocol (dose, route, exposure period) as well as nimal strain used. AGD is an important hormonal-sensible developmental mea- ure which is influenced by endocrinal deregulators, and this arameter has being commonly used to verify sexual dimorphism hanges in rodents. Although it has been demonstrated a possible number of animals/group. CTR = saline, FLX = Fluoxetine 5 mg/kg. There were no estrogenic activity of FLX in immature rat uterotrophic assay [28], the present work did not found any statistical difference in AGD of female offspring from both groups at different ages. The activation and coordination of hypothalamic-pituitary- gonadal (HPG) axis is required to the puberty establishment [29], which is the acquisition of reproductive capability. Some authors state that puberty onset occurs when the gonadotropin release hormone (GnRH) is secreted by hypothalamus [30], while others believe that the first step is the ovary estradiol production and release that will further stimulate the hypothalamic GnRH release [31]. Once released, GnRH will stimulate luteinizing (LH) and folli- cle stimulating (FSH) hormones release by adenohypophysis, which will induce germinative follicles growth and maturation, leading to ovulation and ovarian steroidogenesis. The enhanced estradiol serum levels leads to vaginal opening (VO) and estrous cyclic- ity, which are parameters used to assess puberty establishment. Herein, the in utero and lactational FLX exposure delayed VO and the first estrus of female offspring. The hypothalamic neurotransmission systems regulate the puberty onset during prepubertal period by stimulatory activity ductiv o n e t g a H d e 3 d [ r [ r i s n a o b 5 a t i m t o e r t t g i b d 5 ( e o l s t b m H s t p t t t e o A f d r c t e a A.H. dos Santos et al. / Repro n GnRH, LH and FSH release [31,32]. Cell bodies of serotoninergic eurons are found in raphe nuclei and emit projections to sev- ral brain regions [33], including hypothalamus [34]. It is known hat hypothalamic GnRH-producing neurons receive serotoniner- ic innervation and this neurotransmitter is found in hypophysis nd ovaries [29]. In this way, some studies have evidenced that 5- T plays an important role in the regulation and control of HPG axis ue to its influence on gonadotropin release [35]. Hence, Moran t al. observed that 5-HT subcutaneous administration (25 and 7.5 mg/kg) from PND 30 until the VO day in prepubertal rats, elayed VO, first estrus and reduced the estradiol plasmatic levels 29]. Furthermore, lesions on the dorsal raphe nucleus of female ats in prepubertal period induced a delay in the puberty onset 36]. Thus, 5-HT may have an inhibitory activity on gonadotropins elease, since it was demonstrated that LH release should be inhib- ted by 5-HT neurons from medial raphe nucleus, via GABAergic ystem [37]. On the other hand, 5-HT neurons from dorsal raphe ucleus can stimulate the LH release by a noradrenergic (NA) mech- nism [38] involving locus coeruleus [39]. In the present study, it is suggested that the delay in puberty nset may be due to a delay in estradiol release, which can occur y two mechanisms. The first one considers an inhibitory pattern of -HT on GnRH release, since FLX inhibits 5-HT reuptake, leading to n increase in extracellular concentrations of this neurotransmit- er. This increase promotes stimulation of 5-HT1A somatodendritic nhibitory autoreceptors and 5-HT1D/B presynaptic terminals, cul- inating in reduced 5-HT synthesis and release. However, repeated reatment with FLX promotes downregulation and desensitization f these autoreceptors mechanisms, so 5-HT becomes no longer ffective to inhibit its own release and then the serotoninergic neu- on ceases to be inhibited [40,41]. Furthermore, it has been reported hat repeated exposure to SSRIs could reduce the expression of he 5-HT transporter (SERT), resulting in enhanced serotoniner- ic transmission [42]. Thus, the neuronal impulse flow should be ncreased and the transporters diminished, leading to 5-HT release y axon terminals and this 5-HT can inhibit GnRH secretion. Secon- arily, this inhibitory autoreceptors desensitization enhances the -HT release, leading to a postsynaptic receptors desensitization 5-HT2, 5-HT3, among others) [43]. This second hypothesis should xplain how a serotoninergic stimulatory tone can delay puberty nset, since postsynaptic receptors desensitization would lead to a oss of this excitatory tone on gonadotropin secretion. It is very important to clarify that these effects described above hould not be related to FLX or norfluoxetine plasma concentra- ions, since the exposure was stopped approximately nine days efore the parameters of puberty onset were checked (FLX and its etabolite have a half-life of around 7 and 14.4 h, respectively) [44]. owever, delays in VO and in the first estrus may result from pos- ible neuroadaptations coming from the repeated exposure, since he latency between the end of exposure and the puberty onset arameters assessment may not have been sufficient to normalize he number of receptors as well as its sensitivity [40]. Thus, fur- her investigations are required for a better understanding of how he serotoninergic transmission controls the puberty onset and the strogen release in this phase. Thereby, it is evident the influence of serotoninergic pathways n the HPG axis control during brain development [29,35,45]. lthough the estradiol dosage was not conducted in pubertal emales in the present study, we suggest that 5-HT delays the estra- iol release by ovaries due to its influence on the gonadotropins elease and consequently delayed sexual maturity. This hypothesis ould be strengthened with Matagne et al., which demonstrated hat estradiol administration anticipated the VO date and first strus in prepubertal rats [46]. Although there was a delay in puberty onset, all the parameters nalyzed in these adult females have not changed. This result may e Toxicology 62 (2016) 1–8 7 be due to the fact that 5-HT system modulation on gonadotropin secretion is controversial (some studies showed an inhibitory [36,47] or stimulatory pattern [35,47] or even the absence of activ- ity [35,48] and varies with the animal’s age—prepubertal [35,39,47], peripubertal [35,48] and adult [35,47]. To evaluate these results it is important to consider that VO and first estrus were assessed in prepubertal/pubertal periods while the estrous cycle was evaluated in adult life and the 5-HT influence on gonadotropic hormones release is not the same in both phases. In this way, it should be emphasized that the delay in puberty onset can be due to the proximity between the end of treatment and the pubertal period in which the parameters were evaluated (the interval was 9 days). Thus, it can be assumed that even though FLX and its active metabolite are no longer present in circulating plasma levels (since they have a half-life of around 7 and 14.4 h, respectively) [44], the neuroadaptations resulting from repeated administration can still be present (e.g. receptor desensitization) [40], as well as 5-HT central levels may not yet returned to normal. In contrast, the estrous cycle was assessed in adult females (PND 75), 54 days after the end of FLX exposure. Likewise, the estradiol plasmatic levels, ovarian follicular quantification and uterine morphometric analysis, body weight, reproductive organs weight, sexual and maternal behaviors and fertility test from adult female rats of FLX group were not changed when compared to CTR group. Therefore, it’s suggested that although 5-HT has an effect on puberty onset, it did not have deleterious effects on neuroendocrine and reproductive systems of females in adulthood. 5. Conclusion The present study revealed that in utero and lactational exposure to a human-relevant dose of FLX delays puberty onset in female rats’ offspring. Since a single dose was evaluated, a no-observed adverse effect level could not be determined. The delay in sexual maturity could be related with the 5-HT influences on gonadotropin secretion and reinforces the relevance of further investigations into the mechanisms by which 5-HT acts on HPG axis, controls the GnRH release and delays puberty onset. 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http://refhub.elsevier.com/S0890-6238(16)30056-9/sbref0235 http://refhub.elsevier.com/S0890-6238(16)30056-9/sbref0235 http://refhub.elsevier.com/S0890-6238(16)30056-9/sbref0235 http://refhub.elsevier.com/S0890-6238(16)30056-9/sbref0235 dx.doi.org/10.1159/000124064 dx.doi.org/10.1159/000124064 dx.doi.org/10.1159/000124064 dx.doi.org/10.1159/000124064 dx.doi.org/10.1159/000124064 dx.doi.org/10.1159/000124064 dx.doi.org/10.1159/000124064 In utero and lactational exposure to fluoxetine delays puberty onset in female rats offspring 1 Introduction 2 Material and methods 2.1 Animals and treatment 2.2 Parameters analyzed in female offspring during development (PND 0–60) 2.2.1 Body weight 2.2.2 Physical sexual development 2.3 Parameters analyzed in female offspring during adulthood 2.3.1 Estrous cycle evaluation 2.3.2 Collection of tissues and organs 2.3.3 Plasmatic estradiol quantification 2.3.4 Sexual behavior evaluation 2.3.5 Maternal behavior 2.3.5.1 Maternal behavior evaluation after pup removal 2.3.5.2 Undisturbed mother-pup interaction evaluation 2.3.6 Fertility test 2.4 Statistical analysis 3 Results 3.1 Parameters analyzed in dams 3.1.1 Body weight 3.2 Parameters analyzed in female offspring during development (PND 0–60) 3.2.1 Body weight and physical development 3.2.2 Physical sexual development 3.3 Parameters analyzed in female offspring during adulthood (PND 75-LD 5) 3.3.1 Estrous cycle and estradiol plasmatic concentration 3.3.2 Reproductive organs weight and histology 3.3.3 Sexual behavior 3.3.4 Maternal behavior 3.3.4.1 Maternal behavior evaluation after pup removal 3.3.4.2 Undisturbed mother-pup interaction evaluation 3.3.5 Fertility test 4 Discussion 5 Conclusion Conflict of interest Transparency document References