Perinatal exposure to fluoxetine via placenta and lactation inhibits the testicular development in male rat offspring

Abstract

Due to the widespread use of fluoxetine to treat depression, including pregnant and nursing women, the present study aimed to investigate the effects of in utero and lactational exposure to fluoxetine in rat offspring at post natal day 22. Wistar rat dams were orally treated with fluoxetine (5, 10, and 20 mg/kg) from day 13 gestation to day 21 lactation. Exposure to 10 and 20 mg/kg fluoxetine reduced the body and testis weights. The volume of the seminiferous tubules and epithelium were also reduced following 20 mg/kg fluoxetine exposure. The length of the seminiferous tubules and the population of Sertoli cells changed in offspring exposed to fluoxetine. The amount of seminiferous tubules lacking tubular lumen was higher in rats exposed to 20 mg/kg fluoxetine. Plasma testosterone showed no significant change. In conclusion, fluoxetine exposure via the placenta and lactation may inhibit and delay testicular development, adversely affecting several testicular parameters important for the establishment of sperm production in adulthood.

Keywords:

• fluoxetine
• Leydig cell
• serotonin
• Sertoli cell
• testis

Introduction

Depression is a common psychiatric disorder that affects 10 - 25% of women worldwide. Of particular concern is antepartum and postpartum depression, this condition affects approximately 10 - 15% of childbearing women. Frequently, pharmacological treatment of peripartum depression is advisable because this condition presents risks for both the mother and the infant. There is strong evidence of adverse effects due to maternal depression on cognitive, motor, and emotional development of neonates [Kim et al. 2005]. Cooper et al. [2007] observed that 8.7% of pregnant women were exposed to an antidepressant and 6.2% were exposed to a selective serotonin reuptake inhibitor (SSRI). These authors also reported a great need for further studies to better understand the fetal consequences of exposure to antidepressants.

Several studies have shown adverse effects in the offspring of mothers treated with antidepressants, including SRRIs. Depressed mothers perinatally treated with SSRI showed an increased risk of low birth weight and respiratory distress [Oberlander et al. 2006]. Fetal neurobehavioral development also changes in women treated with SSRI throughout gestation [Mulder et al. 2011]. Developmental exposure to SSRI also induces long-term changes in the hypothalamic-pituitary-adrenal system [Pawluski et al. 2012]. Exposure to SSRI during neonatal life also reduced male sexual behavior [Harris et al. 2012]. Human and animal studies suggest age-dependent effects of SSRIs, with both negative and positive effects during life [Olivier et al. 2011]. According to Homberg et al. [2010], information about adverse effects of SSRI use during pregnancy or the postpartum period is limited and one cannot conclude whether SSRI use for pregnant women is safe or should be discontinued during pregnancy.

Fluoxetine is a selective serotonin (5-hydroxytryptamine, 5HT) reuptake inhibitor largely used to treat depression, including in pregnant and nursing women. It is the second most frequently used antidepressant [Ververs et al. 2006]. In humans, fluoxetine is N-methylated to norfluoxetine, which displays similar anti-depressant properties [Kristensen et al. 1999]. However, due to their high liposolubility, fluoxetine and norfluoxetine can readily cross the placenta and be transferred into the milk during both organogenesis and fetal growth stages [Pohland et al. 1989]. Moreover, the long half-life of these compounds can contribute to a sustained exposure of fetuses and neonates [Heikkinen et al. 2003]. Possible adverse effects of these drugs have been studied in rat offspring treated during pregnancy, lactation, or early postnatal life [Pereira et al. 2007; Silva Junior et al. 2008].

Pharmacological data demonstrated that an increase in brain serotonin can alter the release of luteinizing hormone (LH) and follicle stimulating hormone (FSH) through inhibition of gonadotrophin releasing hormone (GnRH) secretion by the hypothalamus and, therefore, impair steroidogenesis and sperm production in rats [Das et al. 1985]. Also, Shishkina and Dygalo [2000] demonstrated that 5HT could both inhibit and facilitate the modulation of gonadotrophin secretion, depending on the age and sex of the animal. Furthermore, the inhibition of 5HT synthesis in male rats decreases the endocrine function of testis [Aragón et al. 2005].

Considering that developing fetuses can be exposed to fluoxetine and its active metabolite norfluoxetine via placenta and lactation, it is critical to further investigate the effects of these drugs on gonadal development. Therefore, the present study aimed to investigate the effects of in utero and lactational exposure to fluoxetine on several testicular endpoints in rat offspring at post natal day 22.

Results

Weight development of male offspring

The effects of fluoxetine administration on offspring post natal body weight development are shown in Figure 1. Body weight at birth was significantly reduced in offspring exposed to the highest fluoxetine dose (20 mg/kg) when compared to the control and fluoxetine (5 mg/kg) groups. In fact, body weight was persistently reduced in offspring of dams exposed to 20 mg/kg fluoxetine throughout lactation. In addition, offspring exposed to the intermediate dose of 10 mg/kg showed a significant reduction in body weight from post natal day 20 to 22 when compared to controls. On post natal day 22, when offspring were analyzed, body weight of all fluoxetine exposed groups was significantly reduced in relation to the control group (Table 1).

Figure 1.  Postnatal body weight development of male offspring exposed to fluoxetine (5 mg/kg, n = 10; 10 mg/kg, n = 8; 20 mg/kg, n = 10; and control, n = 10) via placenta and lactation.

Table 1. Body, testis, and epididymis weight of 22-day old male offspring exposed to fluoxetine (5 mg/kg, n = 10; 10 mg/kg, n = 8; 20 mg/kg, n = 10; and control, n = 10) via placenta and lactation.

Weight (g)	Control	5 mg/kg	10 mg/kg	20 mg/kg	p
Body	61.6 ± 5.4^a	52.3 ± 6.4^b	49.1 ± 3.6^b	46.6 ± 4.2^b	0.00
Testis	0.14 ± 0.02^a	0.12 ± 0.03^ab	0.10 ± 0.01^bc	0.09 ± 0.00^c	0.00
Epididymis	0.01 ± 0.00	0.02 ± 0.00	0.01 ± 0.00	0.01 ± 0.00	0.17
GSI	0.23 ± 0.02	0.23 ± 0.04	0.22 ± 0.01	0.20 ± 0.02	0.07

GSI: gonadosomatic index; ^a,b,c indicate significant differences.

Results were expressed as mean and (±) standard deviation.

Testis weight reduced in offspring exposed to 10 and 20 mg/kg of fluoxetine when compared to controls (Table 1). The gonadosomatic index (GSI; testis weight corrected for body weight), was of borderline significance (p = 0.07) with an apparent reduction (13%) at fluoxetine levels of 20 mg/kg group. Epididymis weight did not change due to fluoxetine exposure (Table 1).

Volume of testicular components

The volumes of testicular components are shown in Table 2. Fluoxetine treatment at 20 mg/kg reduced the volume of seminiferous tubules, seminiferous epithelium, tubular lumen, and lymphatic space. Significant reductions in the volume of Leydig cells were observed in all fluoxetine exposed groups. However, the volumes of basal membrane, connective tissue, and blood vessels did not change with fluoxetine exposure.

Table 2. Volume of different testicular components of 22-day old male offspring exposed to fluoxetine (5 mg/kg, n = 10; 10 mg/kg, n = 8; 20 mg/kg, n = 10; and control, n = 10) via placenta and lactation.

Volume (μL)	Control	5 mg/kg	10 mg/kg	20 mg/kg	p
Seminiferous tubule	117.2 ± 19.3^a	100.1 ± 25.7^ab	87.8 ± 8.6^ab	76.6 ± 6.3^b	0.00
Seminiferous epithelium	96.4 ± 17.8^a	82.7 ± 22.3^a	69.9 ± 5.3^ab	61.8 ± 4.7^b	0.00
Lumen	8.2 ± 2.0^a	6.2 ± 1.8^ab	5.6 ± 3.4^ab	4.9 ± 1.2^b	0.01
Basal membrane	12.5 ± 2.3	11.0 ± 3.7	12.1 ± 1.6	9.8 ± 1.6	0.09
Leydig cells	5.2 ± 1.2^a	3.0 ± 1.1^b	2.7 ± 1.0^b	2.9 ± 0.8^b	0.00
Connective tissue	0.5 ± 0.2	0.7 ± 0.3	1.1 ± 0.3	0.6 ± 0.2	0.10
Blood vessels	1.8 ± 0.9	2.5 ± 1.6	2.1 ± 0.6	2.1 ± 0.7	0.89
Lymphatic space	7.8 ± 1.5^a	4.3 ± 2.0^b	5.6 ± 1.3^ab	4.5 ± 1.9^b	0.00

^a,b indicate significant differences.

Results were expressed as mean and (±) standard deviation.

Testicular morphometric parameters

Table 3 shows the morphometric results of the testicular parenchyma. Offspring exposed to the highest fluoxetine dose displayed a 27% reduction in the total length of seminiferous tubules when compared to the control group. The Sertoli cell population was reduced 26% in offspring exposed to 20 mg/kg fluoxetine, showing a borderline significance (p = 0.06). Also, the percentage of seminiferous tubules lacking tubular lumen was significantly increased in offspring exposed to this same dose of fluoxetine (Table 3). Tubular diameter and epithelium height did not change due to fluoxetine exposure. Photomicrographs of testicular sections of the control and the fluoxetine exposed rats are shown in Figure 2. Note the absence of pathological lesions.

Figure 2.  Photomicrographs of testicular sections of 22 day old offspring exposed to fluoxetine via placenta and lactation. A) Testicular section of control animal, luminated seminiferous tubules (arrow). B) Testicular section of control rat, luminated seminiferous tubules (ST) and Sertoli cell (arrow). C) Testicular section from fluoxetine 5 mg/kg group, luminated seminiferous tubules (arrow) and intertubular compartment (star). D) Testicular section from fluoxetine 10 mg/kg group, note the germ cells showing morphological characteristics of apoptosis (black arrows) in a seminiferous tubule (ST), Leydig cells (white arrows), and lymphatic space (star). E) Testicular section from fluoxetine 20 mg/kg group, note the presence of luminated seminiferous tubules (white arrows) and tubules undergoing lumen formation (black arrows). F) Testicular section from fluoxetine 20 mg/kg group, note the presence of seminiferous tubules undergoing lumen formation (black arrow) and a luminated seminiferous tubules (white arrows) in greater magnification.

Table 3. Morphometric data and Sertoli cell number of 22-day old male offspring exposed to fluoxetine (5 mg/kg, n = 10; 10 mg/kg, n = 8; 20 mg/kg, n = 10; and control, n = 10) via placenta and lactation.

Parameters	Control	5 mg/kg	10 mg/kg	20 mg/kg	p
Tubular diameter (μm)	147.8 ± 7.2	138.8 ± 24.1	146.16 ± 12.0	141.5 ± 17.2	0.61
Epithelium height (μm)	57.5 ± 3.0	56.0 ± 11.3	56.5 ± 5.4	56.2 ± 5.2	0.96
Sertoli cells/cross section	22.3 ± 2.1	23.4 ± 1.8	23.6 ± 2.2	22.3 ± 2.8	0.52
Seminiferous tubule length (m)	6.8 ± 1.2^a	7.1 ± 2.9^a	5.2 ± 0.6^ab	5.0 ± 1.0^b	0.02
Sertoli cell population (x10^7)	3.8 ± 0.8	4.1 ± 1.7	3.1 ± 0.4	2.8 ± 0.7	0.06
Seminiferous tubules lacking lumen (%)	35.2 ± 5.0^a	34.4 ± 8.5^a	35.6 ± 9.4^ab	48.3 ± 9.7^b	0.02

^a,b indicate significant differences.

Results were expressed as mean and (±) standard deviation.

Leydig cell area and testosterone concentration

No significant differences were observed in the cellular, nuclear, and cytoplasmatic area(s) of Leydig cells for any treatment group (Table 4). In addition, no changes were observed in plasma testosterone (Fig. 3).

Figure 3.  Plasma testosterone levels (ng/mL) of 22 day old offspring exposed to fluoxetine (Flu; 5 mg/kg, n = 10; 10 mg/kg, n = 8; 20 mg/kg, n = 10; and control, n = 10) via placenta and lactation. Results were expressed as mean and (±) standard deviation.

Table 4. Leydig cell cellular, nuclear, and cytoplasmatic area of 22-day old male offspring exposed to fluoxetine (5 mg/kg, n = 10; 10 mg/kg, n = 8; 20 mg/kg, n = 10; and control, n = 10) via placenta and lactation.

Area (µm^2)	Control	5 mg/kg	10 mg/kg	20 mg/kg	p
Cell	161.7 ± 14.0	155.4 ± 21.6	148.6 ± 12.0	140.1 ± 24.0	0.12
Nucleus	52.8 ± 7.7	49.9 ± 6.9	48.1 ± 3.5	46.7 ± 5.8	0.23
Cytoplasm	108.9 ± 12.7	105.5 ± 16.0	100.5 ± 9.4	93.3 ± 18.8	0.17

Results were expressed as mean and (±) standard deviation.

Discussion

Offspring exposed to 20 mg/kg of fluoxetine showed a reduction in body weight at birth. According to Morrison et al. [2005], the acute increase in plasma serotonin observed after fluoxetine administration during gestation may reduce uterine blood flow, which could account for the observed reduction in body weight. In addition, body weight development was significantly impaired throughout lactation in male offspring exposed to fluoxetine. On post natal day 22, a reduction in body weight was observed in all treatment groups. Studies previously demonstrated that intraperitoneal administration of citalopram and fluoxetine can reduce body weight gain of neonate rats [Mendes da Silva et al. 2002; Deiró et al. 2004; Silva Junior et al. 2008]. This change could be related to the inhibitory effect of serotonin on food ingestion [Simansky 1996]. Alternatively, fluoxetine administration may have interfered with intestinal food absorption, as there are reports of partial atrophy of intestinal vilosities following neonatal fluoxetine administration [Macguirk and Siloverstone 1990].

Testis weight was also reduced in offspring exposed to 10 and 20 mg/kg fluoxetine. The gonadosomatic index was reduced by 13% in offspring exposed to the higher dose of fluoxetine. These results are in agreement with the data published by Silva Junior et al. [2008] that showed a 36% reduction in testis weight of neonate rats exposed to 20 mg/kg fluoxetine by the intraperitoneal route.

The testicular parenchyma can be divided into two functional sites, the tubular and the interstitial compartments [França et al. 2005]. In the present study, the tubular compartment was significantly affected by the treatment with 20 mg/kg fluoxetine at post natal day 22, showing a reduction in the volumes of seminiferous tubules, tubular lumen, and seminiferous epithelium. According to França et al. [2000], testis weight is positively correlated with the volume of the tubular components, Sertoli cell population, and daily sperm production. The results obtained in the present study are in agreement with those previously published by Silva Junior et al. [2008], which showed that intraperitoneal administration of fluoxetine to neonate rats reduces testis weight and the volume of tubular compartment components. Adult rats treated with fluoxetine also showed a decrease in thickness of germinal epithelium, diameter of seminiferous tubules, and counts of germ cells [Aggarwal et al. 2012]. Rat dams treated daily by gavage during gestation and lactation with 7.5 mg/kg fluoxetine decrease the number of spermatozoa and reduced seminiferous epithelium height and diameter of seminiferous tubules at post natal day 100 [Vieira et al. 2012]. Thus, the reproductive impairment in adulthood due to fluoxetine treatment also seems to occur via lactation and through the placenta.

A significant reduction in the total volume of Leydig cells were observed in the intersticial compartment, in all treatment groups. This parameter is correlated to Leydig cell number [Silva Junior and França 2001; França et al. 2005]. Accordingly, it is possible that fluoxetine may have reduced the population of Leydig cells during gestation and early life. In rats, fetal Leydig cells are important for pre- and early post natal surges of testosterone, being replaced by adult-type Leydig cells from post natal day 14 onward [Ariyaratne and Mendis-Handagama 2000].

In addition, no significant differences were observed in the Leydig cell cytoplasm or nucleus. The size of Leydig cells and the volume of endoplasmic reticulum are related to the Leydig cell's capability to produce testosterone [França et al. 2005]. These results are in accord with no change in plasma testosterone. In the present study, the reduction in the number of Leydig cells and maintenance of its individual cellular area indicate a probable effect of fluoxetine on the Leydig cell population.

Fluoxetine via the placenta and/or lactation can reduce the volume of seminiferous tubules and epithelium, tubular lumen, length of seminiferous tubules, and the population of Sertoli cells, and increase the amount of seminiferous tubules lacking tubular lumen. These results indicate a delay in testicular development, slowing the transition of non-lumined spermatic cords to lumined seminiferous tubules. The formation of tubular lumen reflects the maturational status of Sertoli cells, which are also responsible for tubular fluid production and the formation of the blood-testis barrier [Orth 1993].

Division and maturation of Sertoli cells, which are responsible for orchestrating the development of the testis in the pre- and early post natal periods, is dependent upon the presence of several endocrine and paracrine signals, including testosterone, FSH, triiodothyronine (T3), and TNF-α [Hellani et al. 2000; Sharpe 2001; Kaitu'u-Lino et al. 2007]. In the present study, the administration of the selective serotonin reuptake inhibitor (fluoxetine) to rat dams may have interfered with gonadotrophin levels in male offspring because serotoninergic neurotransmission can inhibit GnRH release by the hypothalamus [Li and Pelletier 1995]. Considering that the pre- and early post natal periods are critical for testicular development, any hormonal imbalance during this critical window may be harmful for the establishment of sperm output in later life.

We found no previous reports that exposure to 20 mg/kg fluoxetine via the placenta and lactation can impair and delay the testicular development at post natal day 22 (before adulthood). For rats, a dose of 20 mg/kg of fluoxetine per day is considered moderate [Aggarwal et al. 2012]. Furthermore, the effects of maternal treatment with fluoxetine in offspring still need to be better understood in order to provide standard doses for the safe use of fluoxetine. For example, maternal treatment with 7.5 mg/kg fluoxetine during pregnancy and lactation can impair sexual motivation in adult male mice descendants [Gouvêa et al. 2008].

It is of note that in the present study we used healthy dams exposed to fluoextine and not a model of maternal stress to induce depression in dams. Therefore the results may vary and care must be taken when comparing studies.

In conclusion, our results demonstrate that fetal and neonatal exposure of rats to 20 mg/kg fluoxetine reduced the testicular weight, volume and length of seminiferous tubules and the population of Sertoli cells. In addition, a reduction in tubular lumen formation was observed in the offspring exposed to 20 mg/kg fluoxetine, indicating that exposure to fluoxetine via placenta and lactation may inhibit and delay the testicular development in male rat offspring at post natal day 22.

Materials and Methods

Animals and experimental design

Wistar rats were obtained from the vivarium of the Federal Rural University of Pernambuco and kept under controlled temperature (22°C), humidity (50%), and light conditions (12-h light/dark cycle). Standard pellet food (Labina Purina, Paulínia, Brazil) and water were available ad libitum. Twenty pregnant dams were assigned to the following treatment groups (5 dams in each group): I) Control (deionized water); II) fluoxetine chloride 5 mg/kg; III) fluoxetine chloride 10 mg/kg; and IV) fluoxetine chloride 20 mg/kg. Following birth, litter size was standardized to 6 pups. Dams continued to receive the same treatment until weaning (post natal d 21). Male pup controls (n = 10) and pups exposed to fluoxetine (5 mg/kg, n = 10; 10 mg/kg, n = 8; 20 mg/kg, n = 10) via placenta and lactation were then analyzed on post natal d 22.

Dams were treated daily by oral route (gavage) from gestation d 13 to lactation d 21. Fluoxetine chloride was obtained from Roval Manipulation Pharmacy (Recife, Brazil) and diluted in deionized water. Dose levels were based on previous experimental studies with fluoxetine [Gandarias et al. 1999; Frank et al. 2000]. The experimental protocol was approved by the Ethics Committee of the Animal Morphology and Physiology Department of Federal Rural University of Pernambuco (CEEA‐DMFA/UFRPE no. 00014/02) in accordance with the basic principles for research using animals.

Physical development of pups

Body weight development of the male offspring was monitored daily. Testes and epididymis were weighed using a BEL Engineering scale (Mark 500/BRA) with 0.001g precision. The GSI was calculated by the fraction between the medium weights of both testes and the total body weight [Tenorio et al. 2011].

Perfusion and morphometric analysis

Twenty two day old weaned rats were anesthetized (Sodium thiopental 30 mg/kg). Physiological saline solution (NaCl 0.9%) plus sodium heparin (500UI/L) was perfused through the vasculature of each rat via heart for approximately 5 to 10 min. Subsequently, animals were perfused with glutaraldehyde 4% in phosphate buffer 0.01 M pH 7.4 for 20 min. Following perfusion, testes were removed, sectioned in 2 mm thick, dehydrated in graded ethanol solutions, and embedded in glycol methacrylate (Historesin, Leica, Wetzlar, Germany). Sections with a thickness of 4 µm were stained with 1% toluidine blue/sodium borate and analyzed morphologically and morphometrically, as described by Tenorio et al. [2011].

The volume density of each testicular component was obtained using point counting by systematic allocation through a micrometer reticule (U-OCMSQ 10mm/100, Olympus, Tokyo, Japan) with 441 intersection points on histological preparation of the testis at 400x magnification. Fifteen fields were randomly counted, totaling 6,615 points for each animal. The seminiferous tubule volume expressed in µL was established from the product of volume density (%) and testicular liquid weight calculated in milligrams (mg). The value of testicular liquid weight was obtained by subtracting 6.5% (relative to the albuginea) of the testicular gross weight. Testis weight was considered equal to its volume because testicular density is approximately 1.03 to 1.04.

The diameter of 30 randomly selected round seminiferous tubules per animal were obtained using a linear reticule micrometer (U-OCMSQ10/10, Olympus) at 100x magnification. The tubular diameter was performed by means of two diametrically opposite measurements. The total length of seminiferous tubules (TLST) per testis, expressed in meters, was obtained by dividing the seminiferous tubule absolute volume (STAV) by r² (r = diameter / 2) and π value:

Sertoli cells count (SCC) were corrected for nuclear or nucleolar diameter and histological section thickness. The crude counts (CC) were corrected for section thickness (S) and the mean nuclear or nucleolar diameter (ND):

The number of Sertoli cells per testis (NSCT), also called Sertoli cells population, was determined from the corrected count of Sertoli cells per tubule cross section (CSC), section thickness (S), and the total length of seminiferous tubules (TLST):

Leydig cell area analysis

Leydig cell area per cross section was measured in 1,000x magnification. The area of 50 Leydig cells was measured for each animal, chosen randomly. After producing the testicular histological sections, the images capture was performed through an optical microscope (Motic BA300, Hong Kong, China), coupled to a digital camera MOTICAM 2300 (Motic, Hong Kong, China), connected to a microcomputer. Leydig cells cellular, nuclear, and cytoplasmatic area measurement was performed using the biometric software Images Plus 2.0^® (Motic, Hong Kong, China), as previously described by Tenorio et al. [2011].

Plasma testosterone

Blood samples were obtained through puncture of the vein cava in previously heparinized rats (125 UI/100 g). After sampling, blood was centrifuged and plasma was kept at -20^°C until analysis. Plasma samples were analyzed by enzyme immunoassay using a polyclonal anti-testosterone antibody, as previously described by de Siqueira Bringel et al. [2011].

Statistical analysis

The Kolmogorov-Smirnov test was used to check the tendency to normality of the data. For the data considered normally distributed, we used analysis of variance (ANOVA) with Tukey-Kramer post‐hoc test. If the data was not normally distributed, we used the nonparametric test of Kruskal-Wallis with Dunn's post‐hoc test. Data were expressed as mean and (±) standard deviation. Differences were considered statistically significant at p < 0.05.

Declaration of interest: Funding sources supporting the work described in the manuscript: Federal Rural University of Pernambuco, Recife, Pernambuco, Brazil and Federal University of Paraná, Curitiba, Paraná, Brazil. The authors report no declarations of interest.

Author contributions: Performed the animals treatment with fluoxetine: WMdO; Prepared the histological sections: IRdS; Performed the histopathological analysis: IRdS, SMdT; Performed the plasma testosterone analysis: RNdM, AMA; Performed the histomorphometrical analyzes: FCLM, BMT; Oversaw all stages of the present study and wrote this manuscript: VAdSJ.

Abbreviations

SSRI:	=	serotonin reuptake inhibitor
5HT:	=	5-hydroxytryptamine
LH:	=	luteinizing hormone
FSH:	=	follicle stimulating hormone
GnRH:	=	gonadotrophin releasing hormone
GSI:	=	gonadosomatic index.