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Fluoxetine and sertraline effects on rat distal cauda epididymis contraction, sperm count and sperm transit time trough epididymis
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Fluoxetine and sertraline effects on rat distal cauda epididymis contraction, sperm count and sperm transit time trough epididymis Mayara Samala Bezerraa,1, Ana Beatriz Melo Martinsa,1, Francisco Mateus Gonçalves Trajanoa, Talles Henrique de Araújo Pontesa, Luana Talinne da Costa Gomesa, Elaine Cristina Gaviolib, Edilson Dantas da Silva Juniora,b,∗ a b
Mode of Drug Action Laboratory, Federal University of Rio Grande do Norte, Natal, Rio Grande do Norte, Brazil Department of Biophysics and Pharmacology, Federal University of Rio Grande do Norte, Natal, Rio Grande do Norte, Brazil
A R T I C LE I N FO
A B S T R A C T
Keywords: Fluoxetine Sertraline Distal cauda epididymis contraction Sperm count Epididymal sperm transit time
Fluoxetine and sertraline are antidepressants drugs capable to impair male fertility by decreasing the number of sperm cells in the ejaculate. However, the mechanism underlying these effects is still not fully understood. It is also reported that alterations in epididymis contraction induced by different drugs affect the number of sperm cells, leading to male fertility alterations. Therefore, this study aimed to investigate if both fluoxetine and sertraline could affect the rat epididymis contraction, altering the sperm transit and/or sperm count trough rat epididymis. In vitro effects of fluoxetine and sertraline (1, 3 and 10 μM) were evaluated in isolated distal cauda epididymis of rats by pharmacological experiments. The effects of long-term treatment with fluoxetine and sertraline (20 mg/kg, i.p., 21 days) were also checked on distal cauda epididymis contractions, serum testosterone levels, sperm production, sperm reserves and sperm transit time trough rat epididymis. In vitro fluoxetine and sertraline (> 3 μM) impaired the contractions induced by KCl, phenylephrine or carbachol although fluoxetine 1 μM potentiate the phenylephrine-induced contractions. Long-term in vivo treatment with fluoxetine and sertraline promoted: (a) an enhancement of rat distal cauda spontaneous contractions; (b) a potentiation of phenylephrine-induced contractions; (c) a decreased in serum testosterone levels; and (d) a diminished daily sperm production, sperm reserves trough epididymis and sperm transit time in rat cauda epididymis. In conclusion, the alteration in the motor activity of epididymis could be associated to the low sperm count in this organ and accelerated transit time trough epididymal cauda of rats.
1. Introduction One class of drugs that deserves attention in terms of its negative effects on male fertility is the selective serotonin reuptake inhibitors (SSRIs). These drugs are widely used for the treatment of depressive disorder because its well-known efficacy and safety (Goodnick and Goldstein, 1998). Among SSRIs, fluoxetine and sertraline represent the most prescribed drugs (Andrade et al., 2004; Coupland et al., 2015), mainly by men of reproductive age (Drobnis and Nangia, 2017). Although fluoxetine or sertraline has been considered effective and safe antidepressants, the use of these drugs can lead to sexual and fertility changes in a significant number of male patients (Cascade et al., 2009). In fact, several studies have shown that the use of these agents may impair ejaculation (Montejo et al., 2001) and reduce quantity/quality
(DNA integrity, motility) of spermatozoa in the ejaculate (Safarinejad, 2008; Tanrikut and Schlegel, 2007). However, the mechanisms behind the effects of fluoxetine or sertraline mainly on sperm quantity/quality are unknown and still open to investigation. An important and putative target for the anti-fertility effects of several drugs, including antidepressants, is the epididymis. The epididymis develops several functions that are essential for male reproduction, such as sperm transport, maturation and storage (Dacheux and Dacheux, 2014). It is well established that alterations on the sperm transit time through the epididymis are able to impair the sperm maturation process and alter the concentration of these cells in epididymis, affecting both the quality and quantity of spermatozoa in the ejaculate (Fernandez et al., 2008; Gil-Guzman et al., 2001). The sperm transit time through epididymis is a result of the contractile activity of
∗
Corresponding author. Department of Biophysics and Pharmacology, Federal University of Rio Grande do Norte, Av. Senador Salgado Filho, s/n Campus Universitário – Lagoa Nova, Natal, 59072-970, RN, Brazil. E-mail address: [email protected] (E.D.d. Silva Junior). 1 Both authors contributed equally. https://doi.org/10.1016/j.ejphar.2019.172774 Received 26 July 2019; Received in revised form 24 October 2019; Accepted 1 November 2019 0014-2999/ © 2019 Published by Elsevier B.V.
Please cite this article as: Mayara Samala Bezerra, et al., European Journal of Pharmacology, https://doi.org/10.1016/j.ejphar.2019.172774
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Fig. 1. Effects of fluoxetine or sertraline 1, 3 and 10 μM pre-incubated for 30 min on rat distal cauda epididymis contractions induced by KCl (Panel A and D, respectively), phenylphrine (Panel B and E, respectively) or carbachol (Panel C and F, respectively). Each point represents Means ± S.E.M. of at least 5 independent experiments performed with tissues from different rats. *P < 0.05 in relation to Emax of control. Table 1 Maximal contractions (Emax) (% KCl contraction) and pEC50 values for phenylephrine or carbachol in the absence (control) or presence of fluoxetine (1, 3 and 10 μM; pre-incubated for 30 min) in rat distal cauda epididymis. Phenylefrine
Control (n = 6) + Fluoxetine 1 μM (n = 6) + Fluoxetine 3 μM (n = 6) + Fluoxetine 10 μM (n = 6) Carbachol Control (n = 6) + Fluoxetine 1 μM (n = 6) + Fluoxetine 3 μM (n = 6) + Fluoxetine 10 μM (n = 6)
Table 2 Maximal contractions (Emax) (% KCl contraction) and pEC50 values for phenylephrine or carbachol in the absence (control) or presence of sertraline (1, 3 and 10 μM; pre-incubated for 30 min) in rat distal cauda epididymis.
Pharmacological parameters
Phenylefrine
Emax
pEC50
119.2 ± 11.5 92.5 ± 10.5 59.6 ± 8.2a 25.2 ± 6.0a
6.0 6.4 5.9 5.2
± ± ± ±
0.09 0.09a 0.04 0.15a
98.8 77.5 50.6 23.2
5.7 5.5 5.3 4.9
± ± ± ±
0.14 0.13 0.12 0.13a
± ± ± ±
9.4 7.1 5.6a 2.2a
Control (n = 6) + Sertraline 1 μM (n = 6) + Sertraline 3 μM (n = 6) + Sertraline 10 μM (n = 6) Carbachol Control (n = 5) + Sertraline 1 μM (n = 5) + Sertraline 3 μM (n = 5) + Sertraline 10 μM (n = 5)
Pharmacological parameters Emax
pEC50
115.0 ± 8.3 91.5 ± 7.5 73.9 ± 6.2a 34.4 ± 5.2a
6.2 6.2 6.0 5.5
± ± ± ±
0.09 0.10 0.09 0.10a
93.8 70.7 46.7 19.3
5.9 5.5 5.5 5.2
± ± ± ±
0.13 0.07 0.10 0.10a
± ± ± ±
11.6 8.9 7.6a 1.3a
Emax (maximal contraction) expressed in % of KCl contraction and pEC50 (potency, measured as the negative log of EC50) obtained from the nonlinear regression curves shown in Fig. 1B and C. n, number of experiments. a P < 0.05 in relation to control.
Emax (maximal contraction) expressed in % of KCl contraction and pEC50 (potency, measured as the negative log of EC50) obtained from the nonlinear regression curves shown in Fig. 1E and F. Values are Means ± S.E.M. n, number of experiments. aP < 0.05 in relation to control.
epididymal smooth muscle cells which is modulated by androgens, autacoids and autonomic neurotransmitters (Chaturapanich et al., 2002; Elfgen et al., 2018; Ricker, 1998; Sujarit and Pholpramool, 1985). Several studies have demonstrated that both fluoxetine and sertraline presented off-target effects that are able to inhibit smooth muscle contraction in different tissues (Pedroso et al., 2017; Ungvari et al., 2000), alter the balance of autonomic nervous system (Hong et al.,
2017; Shores et al., 2001; Tiradentes et al., 2014) and decrease testosterone levels in rodents or cell lines (Camara et al., 2019; Hansen et al., 2017). In this context, we hypothesized that aforementioned effects for fluoxetine or sertraline could also affect the contractile activity of epididymis, leading to alterations in sperm transit through this organ and, consequently, altering the quality and quantity of sperm cells in the ejaculate. 2
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intraperitoneal route of administration and treatment duration (21 days) for fluoxetine or sertraline were chosen according to previous studies that evaluate the effects of these drugs on genitourinary tract of rodents (Busch et al., 1999; Canpolat et al., 2016; Ozyavuz et al., 2004; Pedroso et al., 2017). Additionally, these doses of fluoxetine and sertraline are known to induce antidepressant-like effects in rodents (Detke et al., 1995). The animals were weighed daily and euthanized for experiments 24 h after the last drug injection. All animals were euthanized between 8:00–9:00 a.m. Blood samples from animals of all groups were collected for testosterone measurements, reproductive organs were also isolated and weighted, distal cauda of epididymis used for pharmacological experiments and the sperm cells recovered from testis or epididymis for analyzing of sperm production, sperm reserves and sperm transit time trough epididymis. 2.2. Rat distal cauda epididymis isolation for organ bath studies The animals were euthanized by decapitation, the whole epididymis was exposed and carefully excised and ~1.5 cm of distal cauda (site 7) (Hinton et al., 1979) were isolated and mounted in a standard a 10 ml organ bath under 1.0 g tension containing a physiological salt solution with the following composition (mM): 138.0 NaCl; 5.7 KCl; 1.8 CaCl2·2H2O; 15.0 NaHCO3; 0.36 NaH2PO4·H2O, and 5.5 glucose, prepared in glass distilled deionized water, bubbled with air, and maintained at 32 °C, pH 7.4. Changes in isometric tension in the rat distal cauda epididymis were recorded with a force displacement transducer (Ugo Basile, Italy) coupled to a Gemini two-channel physiographic recorder (Ugo Basile, Italy). After a 30 min of stabilization period, the tissues were challenged with KCl 80 mM for 5 min to evaluate tissue viability and maximal response stabilization. Thus, the preparation was washed out and after 40 min the experiments were performed.
Fig. 2. Variation of weight (g) from rats submitted to the in vivo treatment with drug-free vehicle (control; n = 14), fluoxetine (n = 15) or sertraline (n = 15) 20 mg/kg for 21 days. Each point represents Means ± S.E.M. of at least 14 different animals.
Therefore, this study was carried out in order to evaluate the effects of in vitro fluoxetine or sertraline on contractions of rat distal cauda epididymis induced by KCl or autonomic drugs such as phenylephrine (adrenergic drug) or carbachol (cholinergic drug). We also assessed the effects of in vivo chronic treatment with both drugs on spontaneous or drugs-induced contractions of rat distal cauda epididymis as well as testosterone levels, sperm production, transit time and sperm reserves through epididymis.
2.2.1. Analyses of in vitro effects of fluoxetine and sertraline on rat distal cauda epididymis contraction Untreated animals were euthanized and distal cauda epididymis isolated and mounted in an organ bath as described above. After checking the tissue viability with KCl and an equilibration period of 40 min, the tissues were submitted to time-course responses for KCl 80 mM for 5 min or cumulative concentration–response curves for phenylphrine (agonist of α1-adrenoceptors) or carbachol (agonist of acetylcholine muscarinic receptors) in the absence or presence of three different concentrations of fluoxetine (1, 3 or 10 μM) or sertraline (1, 3 or 10 μM). Each concentration of fluoxetine or sertraline was equilibrated with the tissues for 30–40 min and during intervals of successive curves the preparation was carefully washed out with physiological salt solution. Pharmacological parameters described below were measured from concentrations response curves and used for comparisons between groups.
2. Material and methods 2.1. Animals and treatment Male (60–90 days old/200–300 g) Wistar rats were obtained from the Animal Facility of Bioscience Center from Federal University of Rio Grande do Norte (Natal, Brazil), and maintained under controlled conditions (25 °C, 12/12 h light/dark cycle). All experimental procedures described in this study were approved by the local Ethics Committee for de Use of Experimental Animals of Federal University of Rio Grande do Norte (Protocol number 0058/2018) and are in accordance with the ARRIVE guidelines (Kilkenny et al., 2010). In this study a total number of 68 rats were used. Some animals were euthanized by decapitation (total number of 24 rats) to evaluate the in vitro effects of fluoxetine or sertraline on the contractions of the isolated distal cauda epididymis by pharmacological experiments. Additionally, rats were also divided into three groups, control animals (treated with drug-free vehicle by 21 days; total number of 14 rats), fluoxetine (20 mg/kg, i.p., daily injections for 21 days; total number of 15 rats) and sertraline-treated animals (20 mg/kg, i.p., daily injections for 21 days; total number of 15 rats). The dose,
2.2.2. Evaluation of rat distal cauda epididymis contractions after 21 days treatment with fluoxetine or sertraline Animals were treated with drug-free vehicle, fluoxetine or sertraline for 21 days followed by distal cauda epididymis isolation for organ bath experiments, as described above. After checking tissue viability and 40 min of stabilization period, the spontaneous contractions were
Table 3 Reproductive organs weight for animals submitted to the in vivo treatment with drug-free vehicle (control), fluoxetine or sertraline 20 mg/kg for 21 days. Organ weight Vas deferens (g) Empty seminal vesicle (g) Testis (g) Epididymis (g)
Control
Fluoxetine 20 mg/kg a
0.045 ± 0.002 (9) 0.32 ± 0.02a (8) 1.34 ± 0.03a (9) 0.44 ± 0.009a (8)
0.074 ± 0.002 (9) 0.45 ± 0.18 (9) 1.55 ± 0.017 (9) 0.50 ± 0.009 (6)
Values are Means ± S.E.M. (number of experiments). aP < 0.05 in relation to control. 3
Sertraline 20 mg/kg 0.046 ± 0.002a (9) 0.27 ± 0.19a (8) 1.36 ± 0.03a (9) 0.33 ± 0.016a (7)
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Table 4 Testis and epididymal sperm parameters from rats submitted to the in vivo treatment with drug-free vehicle (control), fluoxetine or sertraline 20 mg/kg for 21 days. Parameters
Groups Control (n = 6)
Testis Number of spermatids, x106/testis Number of spermatids, x106/g/testis Daily sperm production Caput/corpus of epididymis Sperm number, x106/organ Sperm number, x107/g/organ Sperm transit time (days) Cauda of epididymis Sperm number, x106/organ Sperm number, x107/g/organ Sperm transit time (days)
Fluoxetine 20 mg/kg (n = 6)
Sertraline 20 mg/kg (n = 7)
250.4 ± 20.6 293.8 ± 57.75 41.0 ± 3.37
a
155.8 ± 20.2 128.6 ± 14.7a 25.5 ± 3.31a
167.7 ± 14.9a 150.7 ± 13.3a 27.5 ± 2.44a
90.4 ± 20.8 43.4 ± 7.05 2.17 ± 0.36
32.2 ± 4.56a 18.07 ± 1.54a 1.72 ± 0.36
44.15 ± 4.06a 18.66 ± 2.36a 1.65 ± 0.16
163.6 ± 16.7 63.4 ± 5.90 4.12 ± 0.51
54.71 ± 5.89a 24.09 ± 2.64a 2.39 ± 0.42a
58.13 ± 6.48a 24.91 ± 3.02a 2.21 ± 0.29a
Values are Means ± S.E.M. n, number of experiments. aP < 0.05 in relation to control.
2.4. Sperm production, sperm reserves and sperm transit time trough epididymis The testis from control, fluoxetine or sertraline-treated animals were extracted, decapsulated, weighed, and then homogenized in 5 ml of NaCl 0.9% containing Triton X 100 0.5%. Thereafter, the homogenates were sonicated for 30 s followed by a 10-fold dilution. One sample was transferred to Neubauer chambers (four fields per animal), and homogenization-resistant testicular spermatids (stage 19 of spermiogenesis) were counted. In order to calculate the daily sperm production (DSP), the number of spermatids (per testis) at stage 19 was divided by 6.1, which corresponds to the number of days these spermatids are found in the seminiferous epithelium. In the same way, caput/corpus and cauda epididymis from control, fluoxetine or sertraline-treated animals were cut into small tissue fragments with scissors and then homogenized. Thereafter, sperm cells were counted as described early for the testis. Sperm transit time through the caput/corpus and cauda epididymis was determined by dividing the number of spermatozoa present in each of these regions by DSP (da Silva Junior et al., 2014; Vendramini et al., 2014). 2.5. Pharmacological parameters The pharmacological parameters Emax (maximum contraction) (Jurkiewicz and Jurkiewicz, 1976) and pEC50 (potency), measured as the negative log of EC50 (Jurkiewicz et al., 1977), were determined to allow comparisons between curves of phenylephrine or carbachol.
−1
Fig. 3. Serum testosterone levels (ng.dl ) from rats submitted to the in vivo treatment with drug-free vehicle (control; n = 5), fluoxetine (n = 6) or sertraline (n = 6) 20 mg/kg for 21 days. Each point represents Means ± S.E.M. of at least 5 different animals. *P < 0.05 in relation to control.
2.6. Data and statistical analysis recorded for 10 min for measurements of frequency and amplitude of those contractions. Thereafter, the preparation was washed out and after 40 min, cumulative concentration response curves for phenylephrine or carbachol were obtained. Pharmacological parameters were measured from concentration response curves for comparisons between groups.
The data were calculated as a percentage of the maximum response attained in the preparation, using KCl 80 mm (% KCl contraction) or as gram of tension (g). Curve fitting by non-linear regression for the calculation of Emax or pEC50 was performed with Prism v.5 software (San Diego, CA, USA). Values are presented as means ± standard error of mean (S.E.M.). One-way ANOVA followed by the Dunnet's posttest was used. A p value of less than 0.05 was considered to be statistically significant. The results were obtained from groups of at least five experiments with different tissues from distinct animals.
2.3. Serum testosterone determination At the end of treatment, blood samples were obtained from control and treated animals. Then, serum was obtained by centrifugation and frozen until the moment of hormone determination. Serum testosterone levels were determined by chemiluminescence immunoassay (Access Testosterone kit, Beckman Coulter), with a sensitivity of 10 ng/dl. The analysis was performed at DNA Center Laboratory (Natal, Brazil).
2.7. Drugs and reagents Phenylephrine, carbachol, fluoxetine (for in vitro experiments) and sertraline (for in vitro experiments) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Fluoxetine and Sertraline used for the in vivo treatments were from Viafarma (Brazil) Iberoquímica Farmacêutica (Brazil), respectively. All reagents used for physiological salt solution were from Dinâmica (Brazil). 4
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Fig. 4. Effects of in vivo treatment with drug-free vehicle (control), fluoxetine or sertraline 20 mg/kg for 21 days on spontaneous contractions of rat distal cauda epididymis. Panel A - Original tracings showing the spontaneous contractions of distal cauda epididymis from control, fluoxetine or sertraline treated animals. Panel B and C – Bar graphs showing the effects of in vivo treatment with drugfree vehicle (control), fluoxetine or sertraline 20 mg/kg for 21 days on frequency (Panel B) and amplitude (Panel C) of spontaneous contractions of rat distal cauda epididymis. Each bar represents the means ± S.E.M. of 8–10 independent experiments performed with tissues from different rats. *p < 0.05 in relation to the control.
Fig. 5. Effects of in vivo treatment with drug-free vehicle (control), fluoxetine or sertraline 20 mg/kg for 21 days on rat distal cauda epididymis contractions induced by KCl (Panel A), phenylphrine (Panel B) or carbachol (Panel C). Each point represents Means ± S.E.M. of 8–10 independent experiments performed with tissues from different rats.
3. Results
decreased the KCl-induced distal cauda epididymis contraction by about 30 and 70%, respectively (Fig. 1A). The pre-incubation of fluoxetine 3 and 10 μM were also able to significantly diminish the Emax for phenylephrine (~50 and ~80%, respectively) (Fig. 1B; Table 1) or carbachol (~50 and ~75%, respectively) (Fig. 1C; Table 1). Thus, we also found that phenylephrine was 3-fold more potent in tissues from rats pre-exposed to fluoxetine 1 μM (Fig. 1B; Table 1). On the other hand, phenylephrine and carbachol were 5.5-fold and 7.5-fold,
3.1. In vitro effects of fluoxetine or sertraline on rat distal cauda epididymis contractions The Fig. 1 shows that fluoxetine or sertraline at concentrations higher that 3 μM induced a non-specific reduction in the contractile effects of KCl, phenylephrine or carbachol. Fluoxetine 3 and 10 μM 5
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0.31 ± 0.15, n = 10; Fluoxetine: 2.27 ± 0.48, n = 8; P < 0.05) and amplitude (Control: 0.026 ± 0.015, n = 10; Fluoxetine: 0.15 ± 0.02, n = 8; P < 0.05) of spontaneous contractions of rat distal cauda epididymis while sertraline treatment increased the amplitude (Control: 0.026 ± 0.015, n = 10; Sertraline: 0.11 ± 0.02, n = 8; P < 0.05) without affecting the frequency (Control: 0.31 ± 0.15, n = 10; Sertraline: 0.95 ± 0.28, n = 8; P > 0.05) (Fig. 4). The effects of fluoxetine or sertraline in vivo treatment were also checked on the contractions of rat distal cauda epididymis induced by the depolarizing agent KCl, phenylephrine (agonist of α1-adrenoceptors) or carbachol (agonist of muscarinic receptors) (Fig. 5). The KClinduced distal cauda epididymis contractions were not altered by the in vivo treatment with fluoxetine of sertraline (Fig. 5A). On the other hand, phenylephrine-induced contractions were 3- and 2.3-fold more potent in distal cauda epididymis from fluoxetine and sertraline-treated animals, respectively (Fig. 5B; Table 5). The potency for carbachol was unchanged after treatment with both fluoxetine or sertraline (Fig. 5C, Table 5). Furthermore, no significant changes were found in the maximum effects (Emax) for phenylephrine or carbachol in the treated groups when compared to control (Fig. 5C; Table 5).
Table 5 Maximal contractions (Emax) (% KCl contraction) and pEC50 values for phenylephrine or carbachol in rat distal cauda epididymis from rats submitted to the in vivo treatment with drug-free vehicle (control), fluoxetine or sertraline 20 mg/kg for 21 days. Pharmacological parameters Phenylefrine
Emax
pEC50
Control (n = 10) Fluoxetine 20 mg/kg (n = 8) Sertraline 20 mg/kg (n = 9) Carbachol Control (n = 10) Fluoxetine 20 mg/kg (n = 8) Sertraline 20 mg/kg (n = 10)
0.63 ± 0.03 0.60 ± 0.04 0.74 ± 0.05
6.16 ± 0.09 6.60 ± 0.08a 6.46 ± 0.05a
0.57 ± 0.03 0.55 ± 0.05 0.70 ± 0.05
5.81 ± 0.07 6.05 ± 0.08 5.75 ± 0.06
Emax (maximal contraction) expressed in g of tension and pEC50 (potency, measured as the negative log of EC50) obtained from the non-linear regression curves shown in Fig. 5B and C. Values are Means ± S.E.M. n, number of experiments. aP < 0.05 in relation to control.
respectively, less potent in the presence of fluoxetine 10 μM (Fig. 1B; Table 1). Sertraline 3 and 10 μM also decreased the contractions of rat distal cauda epididymis induced by a single concentration of KCl by about 40 and 75%, respectively (Fig. 1D). The pre-incubation of sertraline 3 and 10 μM depressed the Emax for phenylephrine (~35 and ~70%, respectively) (Fig. 1E; Table 2) or carbachol (~50 and ~80%, respectively) (Fig. 1F; Table 2). The phenylephrine and carbachol potency was decreased by 3- and 5-fold, respectively, after pre-incubation of sertraline 10 μM (Fig. 1E and F; Table 2). Sertraline 1 μM did not induce any alterations in the contractions of rat distal cauda epididymis induced by KCl, phenylephrine or carbachol (Fig. 1; Table 2).
4. Discussion This study showed that in vitro fluoxetine or sertraline (> 3 μM) impaired the contractions induced by KCl or exogenous agonists although fluoxetine 1 μM potentiate the phenylephrine-induced rat distal cauda epididymis contractions. Furthermore, in vivo treatment with fluoxetine and sertraline decreased daily sperm production, sperm reserves trough epididymis and sperm transit time in cauda epididymis in the rats. Thus, these aforementioned in vivo effects found in treated animals could be attributed to the reduction of serum testosterone levels, the enhancement of spontaneous contractions or potentiation of adrenergic agonist-induced distal cauda epididymis contractions. The motor activity of smooth muscle cells from epididymal duct is an important process to the transport of spermatozoa within the epididymis. It is widely accepted that during these transit trough epididymis, spermatozoa undergo maturation, acquire motility and fertilizing capacity (Dacheux and Dacheux, 2014; Elfgen et al., 2018). Several mechanisms may be important to the regulation of smooth muscle function in epididymis such as neuronal input (adrenergic and cholinergic input), hormonal, epithelial and sperm or luminal factors (Elfgen et al., 2018). Thus, any drug that interferes in smooth muscle contractility or its regulation process might affect the epididymis contraction, preventing the correct maturation and transport of sperm (da Silva Junior et al., 2014; Elfgen et al., 2018). It has been reported that both fluoxetine and sertraline negatively affect the smooth muscle contraction. For instance, fluoxetine or sertraline > 3 μM reduced the contractions induced by KCl or adrenergic agonists in rodent (Busch et al., 2000; Kalyoncu et al., 1999; Pedroso et al., 2017) or human vas deferens (Medina et al., 2000). Sertraline also inhibited the acetylcholine-induced guinea pig bladder contractions (Uno et al., 2017) and relaxed the pre-contracted human isolated mesenteric arteries (Vila et al., 1999). Therefore, we hypothesized that both fluoxetine and sertraline could also affect rat epididymis contraction. In our study, both fluoxetine and sertraline (> 3 μM) presented an inhibitory effects on KCl, phenylephrine or carbachol induced contractions, as characterized by a significant decrease in the maximum effect. This inhibitory effect could be attributed to the blockade of calcium channels, as it has been argued by previous studies (Busch et al., 2000; Kalyoncu et al., 1999; Medina et al., 2000; Pedroso et al., 2017; Ungvari et al., 2000; Uno et al., 2017). In fact, Deak et al. (2000) using patch-clamp technique in the whole-cell configuration reported voltage-gated calcium channels were inhibited by fluoxetine. Similarly, it is also described that sertraline presented the same pharmacological behavior (Lee et al., 2012). This antagonistic effect of fluoxetine and
3.2. Effects of chronic treatment with fluoxetine or sertraline on animals and reproductive organ weight Animals treated with drug-free vehicle presented a weight gain of 73.57 g ± 5.17 (n = 12) after 21 days of the treatment while fluoxetine and sertraline-treated rats showed a significant weight loss (P < 0.05 compared to control) of −54.45 g ± 6.56 (n = 11) and −38.8 g ± 13.41 (n = 12), respectively (Fig. 2). The in vivo treatment with fluoxetine and sertraline for 21 days also reduced the vas deferens, empty vesicle seminal, testis and epididymis weight when compared to control (Table 3). 3.3. Effects of chronic treatment with fluoxetine or sertraline on rat sperm production, sperm reserves, sperm transit time trough epididymis and testosterone levels The in vivo treatment with fluoxetine or sertraline for 21 days decreased the number of spermatids in the rat testis and the daily sperm production (DSP) as well (Table 4). A significant decrease was also found in the sperm reserves in caput/corpus and cauda of epididymis from fluoxetine and sertraline-treated animals. Furthermore, the treatment with fluoxetine or sertraline accelerated the sperm transit time trough rat cauda epididymis while sperm transit time of caput/body epididymis was unaltered (Table 4). We also found a decrease in serum testosterone levels in fluoxetine or sertraline-treated animals by about 60 and 55%, respectively, when compared to control (Fig. 3). 3.4. Effect of chronic treatment with fluoxetine or sertraline on rat distal cauda epididymis contractions The Fig. 4 shows the effects of in vivo treatment with fluoxetine or sertraline on spontaneous contractions of rat distal cauda epididymis. Fluoxetine treatment significantly augmented the frequency (Control: 6
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fluoxetine and sertraline accelerated the sperm transit time in rat cauda epididymis, plausibly indicating once more an alteration in motor activity of this epididymal site. In other to check this hypothesis, we assessed the spontaneous and drug-induced rat distal cauda epididymis contractions by functional experiments. Isolated distal cauda epididymis showed an increase in the spontaneous motor activity in treated animals, characterized by an increase in the frequency and/or amplitude of these contractions. The autorhythmicity of epididymal duct contractions are important to provide spermatozoa propulsion through the epididymal duct, ensuring sperm maturation and male fertility (Elfgen et al., 2018; Mewe et al., 2006). This process seems to be originated from epithelial PGF (2alpha)-release (Mewe et al., 2006) and it is under control of several factors, including testosterone (Elfgen et al., 2018). Actually, studies indicated that testosterone has important regulatory effects on smooth muscle contractility of genitourinary tract, as demonstrated by the development of spontaneous contractions in rodent vas deferens or seminal vesicle after castration (Hib et al., 1985; MacDonald and McGrath, 1980), increasing of spontaneous contraction amplitudes in caput/ corpus, and augmented basal intraluminal pressure in corpus and cauda epididymis after castration (Din-Udom et al., 1985). Therefore, we suggest that fluoxetine and sertraline treatment increased the spontaneous contractions, and consequently, the sperm transit time of rat distal cauda epididymis indirectly by diminishing the serum testosterone levels. Epididymal smooth muscle contractions elicited by adrenergic and cholinergic neurotransmitters via α1-adrenoceptors and M3 receptors, respectively, also play a significant role on sperm transport trough epididymis, mainly in epididymal cauda (Dacheux and Dacheux, 2014; Elfgen et al., 2018). Then, we also checked the effects of long-term in vivo treatment with fluoxetine or sertraline on rat distal cauda epididymis contractions induced by phenylephrine (α1-adrenoceptors) or carbachol (muscarinic receptor agonist). Phenylephrine was more potent to induce epididymal contractions in fluoxetine and sertraline treated animals compared with controls, while no significant alterations were found for carbachol or KCl-induced contractions. A potentiation of noradrenaline contractile responses was previously observed in vas deferens from animals chronically treated with fluoxetine (Busch et al., 1999) or sertraline (Ozyavuz et al., 2004). The mechanism underlying the aforementioned effects of fluoxetine or sertraline is not fully understood, but it was proposed that both drugs could induce supersensitivity due to noradrenaline exhaustion caused by prolonged inhibition of noradrenaline uptake system. More studies are needed for a comprehensive view of fluoxetine and sertraline longterm effects on adrenergic neurotransmission of genitourinary tract. However, the potentiation of adrenergic-induced contractions by both drugs could be another important mechanism to accelerate the sperm transit trough rat cauda epididymis.
sertraline on calcium channels seems to play a major role in its inhibitory actions on smooth muscle contraction. Fluoxetine 1 μM potentiated the phenylephrine-induced contractions of rat distal cauda epididymis without affecting the carbachol potency, indicating a selective action on epididymal adrenergic system. Similar observation was reported by Busch et al. (2000) who showed that in vitro fluoxetine increased the contractile effects of low noradrenaline concentrations in rat vas deferens, and such effect was attributed to a putative blockade of noradrenaline reuptake process. Although considered a selective serotonin reuptake inhibitor, it is reported that fluoxetine at high concentrations also inhibits the noradrenaline transporter (estimated KD 1.56 μM) (Wenthur et al., 2014). Considering that phenylephrine is also susceptible to neuronal noradrenaline transporter (8.5-fold less than noradrenaline) (Iversen, 1967), we postulate that the leftward shift of phenylphrine curves in the presence of fluoxetine could be associated to its interference on noradrenaline neuronal uptake system in rat epididymis. Conversely, fluoxetine 10 μM decreased the potency of phenylephrine and carbachol. The rightward shift of phenylephrine or carbachol curves in the presence of fluoxetine could be consequence of its affinity for both α1adrenoceptors (estimated KD 2.26 μM) and M3 receptors (estimated KD 1 μM) (Wenthur et al., 2014). Sertraline 10 μM showed the same pharmacological behavior than fluoxetine 10 μM on the potencies for phenylephrine and carbachol. It is described that sertraline is able to inhibit the α1-adrenoceptors (estimated Ki 36–190 nM) and muscarinic receptors (estimated Ki 200 nM) (Owens et al., 1997; Sanchez et al., 2014). Then, the off-targets effects of sertraline on α1-adrenoceptors and muscarinic receptors could be associated to the reduction of phenylephrine or carbachol potency in the presence of this drug. Sertraline is also able to inhibit the noradrenaline neuronal uptake system (estimated Ki 420–820 nM) (Sanchez et al., 2014), but no potentiation of phenylephrine responses was found in our study. This could be probably due to the relative high affinity of sertraline for α1-adrenoceptors that could counteract its effects on noradrenaline uptake system. It is also noteworthy to cite that the negative contractile effects of both fluoxetine or sertraline (on Emax or potency) in rat epididymis was often found in concentrations higher than 3 μM, and therefore, it is still debatable the therapeutic implication of this off-target effects. We found that long-term in vivo treatment of rats with fluoxetine or sertraline induced a marked decrease in daily sperm production and serum testosterone levels. These results are similar to those reported earlier which showed that both fluoxetine and sertraline decrease androgen production in rodents or cell lines (Camara et al., 2019; Hansen et al., 2017; Jahromy and Moghadam, 2014; Munkboel et al., 2018), leading to a spermatogenesis impairment (Aggarwal et al., 2012; Bataineh and Daradka, 2007; Camara et al., 2019; Erdemir et al., 2014). The exact mechanism underlying the negative effects of fluoxetine or sertraline on testicular steroidogenesis is still under investigation, but recent evidence suggest these drugs are able to disrupt androgen synthesis by interfering on steroidogenic enzymes activity (Hansen et al., 2017; Munkboel et al., 2018). Moreover, the reduction in testosterone levels could also be associated in the decrease of animal and organ weights, although other mechanisms not explored for this study could be involved (Scabia et al., 2018; Silverstein-Metzler et al., 2016). Chronic treatment with fluoxetine or sertraline are able to reduce sperm counts in human ejaculate (Akasheh et al., 2014; Safarinejad, 2008) or rodent cauda epididymis (Atli et al., 2017; Bataineh and Daradka, 2007). Likewise, we also found a decrease in sperm number in rat epididymis after treatment with both SSRIs. This may be a direct consequence of testosterone decreased production or alterations in sperm transport along epididymis, since drugs (i.e. sibutramine, bisphenol-A, diethylstilbestrol, indomethacin etc) that are known to affect steroidogenesis and/or epididymis contractions lead to a sperm count reduction (Bagoji et al., 2017; Bellentani et al., 2011; Cariati et al., 2019; Fernandez et al., 2008). Further, we also found that
5. Conclusion In conclusion, it is clear that in vitro or long-term treatment with fluoxetine and sertraline are able to affect rat epididymal contraction. The alteration in the motor activity of rat epididymis induced by fluoxetine and sertraline could be associated to the low sperm count in this organ and accelerated transit time trough epididymal cauda. Altogether, our data indicate that epididymis might be an important target for the anti-fertility effects of fluoxetine or sertraline. Declaration of competing interest The authors declare no conflict of interest. Acknowledgments Thanks are due to Flávio Maurílio dos Santos Lima, Cesar Augusto 7
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Trevisan Bordignon and Ramon Tadeu Galvão Alves Rodrigues for excellent technical support and to the Pro-Rectory of Research of Federal University of Rio Grande do Norte (Portuguese: Pro-Reitoria de Pesquisa da Universidade Federal do Rio Grande do Norte), CNPq and CAPES for financial support.
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Fluoxetine and sertraline effects on rat distal cauda epididymis contraction, sperm count and sperm transit time trough epididymis Mayara Samala Bezerraa,1, Ana Beatriz Melo Martinsa,1, Francisco Mateus Gonçalves Trajanoa, Talles Henrique de Araújo Pontesa, Luana Talinne da Costa Gomesa, Elaine Cristina Gaviolib, Edilson Dantas da Silva Juniora,b,∗ a b
Mode of Drug Action Laboratory, Federal University of Rio Grande do Norte, Natal, Rio Grande do Norte, Brazil Department of Biophysics and Pharmacology, Federal University of Rio Grande do Norte, Natal, Rio Grande do Norte, Brazil
A R T I C LE I N FO
A B S T R A C T
Keywords: Fluoxetine Sertraline Distal cauda epididymis contraction Sperm count Epididymal sperm transit time
Fluoxetine and sertraline are antidepressants drugs capable to impair male fertility by decreasing the number of sperm cells in the ejaculate. However, the mechanism underlying these effects is still not fully understood. It is also reported that alterations in epididymis contraction induced by different drugs affect the number of sperm cells, leading to male fertility alterations. Therefore, this study aimed to investigate if both fluoxetine and sertraline could affect the rat epididymis contraction, altering the sperm transit and/or sperm count trough rat epididymis. In vitro effects of fluoxetine and sertraline (1, 3 and 10 μM) were evaluated in isolated distal cauda epididymis of rats by pharmacological experiments. The effects of long-term treatment with fluoxetine and sertraline (20 mg/kg, i.p., 21 days) were also checked on distal cauda epididymis contractions, serum testosterone levels, sperm production, sperm reserves and sperm transit time trough rat epididymis. In vitro fluoxetine and sertraline (> 3 μM) impaired the contractions induced by KCl, phenylephrine or carbachol although fluoxetine 1 μM potentiate the phenylephrine-induced contractions. Long-term in vivo treatment with fluoxetine and sertraline promoted: (a) an enhancement of rat distal cauda spontaneous contractions; (b) a potentiation of phenylephrine-induced contractions; (c) a decreased in serum testosterone levels; and (d) a diminished daily sperm production, sperm reserves trough epididymis and sperm transit time in rat cauda epididymis. In conclusion, the alteration in the motor activity of epididymis could be associated to the low sperm count in this organ and accelerated transit time trough epididymal cauda of rats.
1. Introduction One class of drugs that deserves attention in terms of its negative effects on male fertility is the selective serotonin reuptake inhibitors (SSRIs). These drugs are widely used for the treatment of depressive disorder because its well-known efficacy and safety (Goodnick and Goldstein, 1998). Among SSRIs, fluoxetine and sertraline represent the most prescribed drugs (Andrade et al., 2004; Coupland et al., 2015), mainly by men of reproductive age (Drobnis and Nangia, 2017). Although fluoxetine or sertraline has been considered effective and safe antidepressants, the use of these drugs can lead to sexual and fertility changes in a significant number of male patients (Cascade et al., 2009). In fact, several studies have shown that the use of these agents may impair ejaculation (Montejo et al., 2001) and reduce quantity/quality
(DNA integrity, motility) of spermatozoa in the ejaculate (Safarinejad, 2008; Tanrikut and Schlegel, 2007). However, the mechanisms behind the effects of fluoxetine or sertraline mainly on sperm quantity/quality are unknown and still open to investigation. An important and putative target for the anti-fertility effects of several drugs, including antidepressants, is the epididymis. The epididymis develops several functions that are essential for male reproduction, such as sperm transport, maturation and storage (Dacheux and Dacheux, 2014). It is well established that alterations on the sperm transit time through the epididymis are able to impair the sperm maturation process and alter the concentration of these cells in epididymis, affecting both the quality and quantity of spermatozoa in the ejaculate (Fernandez et al., 2008; Gil-Guzman et al., 2001). The sperm transit time through epididymis is a result of the contractile activity of
∗
Corresponding author. Department of Biophysics and Pharmacology, Federal University of Rio Grande do Norte, Av. Senador Salgado Filho, s/n Campus Universitário – Lagoa Nova, Natal, 59072-970, RN, Brazil. E-mail address: [email protected] (E.D.d. Silva Junior). 1 Both authors contributed equally. https://doi.org/10.1016/j.ejphar.2019.172774 Received 26 July 2019; Received in revised form 24 October 2019; Accepted 1 November 2019 0014-2999/ © 2019 Published by Elsevier B.V.
Please cite this article as: Mayara Samala Bezerra, et al., European Journal of Pharmacology, https://doi.org/10.1016/j.ejphar.2019.172774
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Fig. 1. Effects of fluoxetine or sertraline 1, 3 and 10 μM pre-incubated for 30 min on rat distal cauda epididymis contractions induced by KCl (Panel A and D, respectively), phenylphrine (Panel B and E, respectively) or carbachol (Panel C and F, respectively). Each point represents Means ± S.E.M. of at least 5 independent experiments performed with tissues from different rats. *P < 0.05 in relation to Emax of control. Table 1 Maximal contractions (Emax) (% KCl contraction) and pEC50 values for phenylephrine or carbachol in the absence (control) or presence of fluoxetine (1, 3 and 10 μM; pre-incubated for 30 min) in rat distal cauda epididymis. Phenylefrine
Control (n = 6) + Fluoxetine 1 μM (n = 6) + Fluoxetine 3 μM (n = 6) + Fluoxetine 10 μM (n = 6) Carbachol Control (n = 6) + Fluoxetine 1 μM (n = 6) + Fluoxetine 3 μM (n = 6) + Fluoxetine 10 μM (n = 6)
Table 2 Maximal contractions (Emax) (% KCl contraction) and pEC50 values for phenylephrine or carbachol in the absence (control) or presence of sertraline (1, 3 and 10 μM; pre-incubated for 30 min) in rat distal cauda epididymis.
Pharmacological parameters
Phenylefrine
Emax
pEC50
119.2 ± 11.5 92.5 ± 10.5 59.6 ± 8.2a 25.2 ± 6.0a
6.0 6.4 5.9 5.2
± ± ± ±
0.09 0.09a 0.04 0.15a
98.8 77.5 50.6 23.2
5.7 5.5 5.3 4.9
± ± ± ±
0.14 0.13 0.12 0.13a
± ± ± ±
9.4 7.1 5.6a 2.2a
Control (n = 6) + Sertraline 1 μM (n = 6) + Sertraline 3 μM (n = 6) + Sertraline 10 μM (n = 6) Carbachol Control (n = 5) + Sertraline 1 μM (n = 5) + Sertraline 3 μM (n = 5) + Sertraline 10 μM (n = 5)
Pharmacological parameters Emax
pEC50
115.0 ± 8.3 91.5 ± 7.5 73.9 ± 6.2a 34.4 ± 5.2a
6.2 6.2 6.0 5.5
± ± ± ±
0.09 0.10 0.09 0.10a
93.8 70.7 46.7 19.3
5.9 5.5 5.5 5.2
± ± ± ±
0.13 0.07 0.10 0.10a
± ± ± ±
11.6 8.9 7.6a 1.3a
Emax (maximal contraction) expressed in % of KCl contraction and pEC50 (potency, measured as the negative log of EC50) obtained from the nonlinear regression curves shown in Fig. 1B and C. n, number of experiments. a P < 0.05 in relation to control.
Emax (maximal contraction) expressed in % of KCl contraction and pEC50 (potency, measured as the negative log of EC50) obtained from the nonlinear regression curves shown in Fig. 1E and F. Values are Means ± S.E.M. n, number of experiments. aP < 0.05 in relation to control.
epididymal smooth muscle cells which is modulated by androgens, autacoids and autonomic neurotransmitters (Chaturapanich et al., 2002; Elfgen et al., 2018; Ricker, 1998; Sujarit and Pholpramool, 1985). Several studies have demonstrated that both fluoxetine and sertraline presented off-target effects that are able to inhibit smooth muscle contraction in different tissues (Pedroso et al., 2017; Ungvari et al., 2000), alter the balance of autonomic nervous system (Hong et al.,
2017; Shores et al., 2001; Tiradentes et al., 2014) and decrease testosterone levels in rodents or cell lines (Camara et al., 2019; Hansen et al., 2017). In this context, we hypothesized that aforementioned effects for fluoxetine or sertraline could also affect the contractile activity of epididymis, leading to alterations in sperm transit through this organ and, consequently, altering the quality and quantity of sperm cells in the ejaculate. 2
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intraperitoneal route of administration and treatment duration (21 days) for fluoxetine or sertraline were chosen according to previous studies that evaluate the effects of these drugs on genitourinary tract of rodents (Busch et al., 1999; Canpolat et al., 2016; Ozyavuz et al., 2004; Pedroso et al., 2017). Additionally, these doses of fluoxetine and sertraline are known to induce antidepressant-like effects in rodents (Detke et al., 1995). The animals were weighed daily and euthanized for experiments 24 h after the last drug injection. All animals were euthanized between 8:00–9:00 a.m. Blood samples from animals of all groups were collected for testosterone measurements, reproductive organs were also isolated and weighted, distal cauda of epididymis used for pharmacological experiments and the sperm cells recovered from testis or epididymis for analyzing of sperm production, sperm reserves and sperm transit time trough epididymis. 2.2. Rat distal cauda epididymis isolation for organ bath studies The animals were euthanized by decapitation, the whole epididymis was exposed and carefully excised and ~1.5 cm of distal cauda (site 7) (Hinton et al., 1979) were isolated and mounted in a standard a 10 ml organ bath under 1.0 g tension containing a physiological salt solution with the following composition (mM): 138.0 NaCl; 5.7 KCl; 1.8 CaCl2·2H2O; 15.0 NaHCO3; 0.36 NaH2PO4·H2O, and 5.5 glucose, prepared in glass distilled deionized water, bubbled with air, and maintained at 32 °C, pH 7.4. Changes in isometric tension in the rat distal cauda epididymis were recorded with a force displacement transducer (Ugo Basile, Italy) coupled to a Gemini two-channel physiographic recorder (Ugo Basile, Italy). After a 30 min of stabilization period, the tissues were challenged with KCl 80 mM for 5 min to evaluate tissue viability and maximal response stabilization. Thus, the preparation was washed out and after 40 min the experiments were performed.
Fig. 2. Variation of weight (g) from rats submitted to the in vivo treatment with drug-free vehicle (control; n = 14), fluoxetine (n = 15) or sertraline (n = 15) 20 mg/kg for 21 days. Each point represents Means ± S.E.M. of at least 14 different animals.
Therefore, this study was carried out in order to evaluate the effects of in vitro fluoxetine or sertraline on contractions of rat distal cauda epididymis induced by KCl or autonomic drugs such as phenylephrine (adrenergic drug) or carbachol (cholinergic drug). We also assessed the effects of in vivo chronic treatment with both drugs on spontaneous or drugs-induced contractions of rat distal cauda epididymis as well as testosterone levels, sperm production, transit time and sperm reserves through epididymis.
2.2.1. Analyses of in vitro effects of fluoxetine and sertraline on rat distal cauda epididymis contraction Untreated animals were euthanized and distal cauda epididymis isolated and mounted in an organ bath as described above. After checking the tissue viability with KCl and an equilibration period of 40 min, the tissues were submitted to time-course responses for KCl 80 mM for 5 min or cumulative concentration–response curves for phenylphrine (agonist of α1-adrenoceptors) or carbachol (agonist of acetylcholine muscarinic receptors) in the absence or presence of three different concentrations of fluoxetine (1, 3 or 10 μM) or sertraline (1, 3 or 10 μM). Each concentration of fluoxetine or sertraline was equilibrated with the tissues for 30–40 min and during intervals of successive curves the preparation was carefully washed out with physiological salt solution. Pharmacological parameters described below were measured from concentrations response curves and used for comparisons between groups.
2. Material and methods 2.1. Animals and treatment Male (60–90 days old/200–300 g) Wistar rats were obtained from the Animal Facility of Bioscience Center from Federal University of Rio Grande do Norte (Natal, Brazil), and maintained under controlled conditions (25 °C, 12/12 h light/dark cycle). All experimental procedures described in this study were approved by the local Ethics Committee for de Use of Experimental Animals of Federal University of Rio Grande do Norte (Protocol number 0058/2018) and are in accordance with the ARRIVE guidelines (Kilkenny et al., 2010). In this study a total number of 68 rats were used. Some animals were euthanized by decapitation (total number of 24 rats) to evaluate the in vitro effects of fluoxetine or sertraline on the contractions of the isolated distal cauda epididymis by pharmacological experiments. Additionally, rats were also divided into three groups, control animals (treated with drug-free vehicle by 21 days; total number of 14 rats), fluoxetine (20 mg/kg, i.p., daily injections for 21 days; total number of 15 rats) and sertraline-treated animals (20 mg/kg, i.p., daily injections for 21 days; total number of 15 rats). The dose,
2.2.2. Evaluation of rat distal cauda epididymis contractions after 21 days treatment with fluoxetine or sertraline Animals were treated with drug-free vehicle, fluoxetine or sertraline for 21 days followed by distal cauda epididymis isolation for organ bath experiments, as described above. After checking tissue viability and 40 min of stabilization period, the spontaneous contractions were
Table 3 Reproductive organs weight for animals submitted to the in vivo treatment with drug-free vehicle (control), fluoxetine or sertraline 20 mg/kg for 21 days. Organ weight Vas deferens (g) Empty seminal vesicle (g) Testis (g) Epididymis (g)
Control
Fluoxetine 20 mg/kg a
0.045 ± 0.002 (9) 0.32 ± 0.02a (8) 1.34 ± 0.03a (9) 0.44 ± 0.009a (8)
0.074 ± 0.002 (9) 0.45 ± 0.18 (9) 1.55 ± 0.017 (9) 0.50 ± 0.009 (6)
Values are Means ± S.E.M. (number of experiments). aP < 0.05 in relation to control. 3
Sertraline 20 mg/kg 0.046 ± 0.002a (9) 0.27 ± 0.19a (8) 1.36 ± 0.03a (9) 0.33 ± 0.016a (7)
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Table 4 Testis and epididymal sperm parameters from rats submitted to the in vivo treatment with drug-free vehicle (control), fluoxetine or sertraline 20 mg/kg for 21 days. Parameters
Groups Control (n = 6)
Testis Number of spermatids, x106/testis Number of spermatids, x106/g/testis Daily sperm production Caput/corpus of epididymis Sperm number, x106/organ Sperm number, x107/g/organ Sperm transit time (days) Cauda of epididymis Sperm number, x106/organ Sperm number, x107/g/organ Sperm transit time (days)
Fluoxetine 20 mg/kg (n = 6)
Sertraline 20 mg/kg (n = 7)
250.4 ± 20.6 293.8 ± 57.75 41.0 ± 3.37
a
155.8 ± 20.2 128.6 ± 14.7a 25.5 ± 3.31a
167.7 ± 14.9a 150.7 ± 13.3a 27.5 ± 2.44a
90.4 ± 20.8 43.4 ± 7.05 2.17 ± 0.36
32.2 ± 4.56a 18.07 ± 1.54a 1.72 ± 0.36
44.15 ± 4.06a 18.66 ± 2.36a 1.65 ± 0.16
163.6 ± 16.7 63.4 ± 5.90 4.12 ± 0.51
54.71 ± 5.89a 24.09 ± 2.64a 2.39 ± 0.42a
58.13 ± 6.48a 24.91 ± 3.02a 2.21 ± 0.29a
Values are Means ± S.E.M. n, number of experiments. aP < 0.05 in relation to control.
2.4. Sperm production, sperm reserves and sperm transit time trough epididymis The testis from control, fluoxetine or sertraline-treated animals were extracted, decapsulated, weighed, and then homogenized in 5 ml of NaCl 0.9% containing Triton X 100 0.5%. Thereafter, the homogenates were sonicated for 30 s followed by a 10-fold dilution. One sample was transferred to Neubauer chambers (four fields per animal), and homogenization-resistant testicular spermatids (stage 19 of spermiogenesis) were counted. In order to calculate the daily sperm production (DSP), the number of spermatids (per testis) at stage 19 was divided by 6.1, which corresponds to the number of days these spermatids are found in the seminiferous epithelium. In the same way, caput/corpus and cauda epididymis from control, fluoxetine or sertraline-treated animals were cut into small tissue fragments with scissors and then homogenized. Thereafter, sperm cells were counted as described early for the testis. Sperm transit time through the caput/corpus and cauda epididymis was determined by dividing the number of spermatozoa present in each of these regions by DSP (da Silva Junior et al., 2014; Vendramini et al., 2014). 2.5. Pharmacological parameters The pharmacological parameters Emax (maximum contraction) (Jurkiewicz and Jurkiewicz, 1976) and pEC50 (potency), measured as the negative log of EC50 (Jurkiewicz et al., 1977), were determined to allow comparisons between curves of phenylephrine or carbachol.
−1
Fig. 3. Serum testosterone levels (ng.dl ) from rats submitted to the in vivo treatment with drug-free vehicle (control; n = 5), fluoxetine (n = 6) or sertraline (n = 6) 20 mg/kg for 21 days. Each point represents Means ± S.E.M. of at least 5 different animals. *P < 0.05 in relation to control.
2.6. Data and statistical analysis recorded for 10 min for measurements of frequency and amplitude of those contractions. Thereafter, the preparation was washed out and after 40 min, cumulative concentration response curves for phenylephrine or carbachol were obtained. Pharmacological parameters were measured from concentration response curves for comparisons between groups.
The data were calculated as a percentage of the maximum response attained in the preparation, using KCl 80 mm (% KCl contraction) or as gram of tension (g). Curve fitting by non-linear regression for the calculation of Emax or pEC50 was performed with Prism v.5 software (San Diego, CA, USA). Values are presented as means ± standard error of mean (S.E.M.). One-way ANOVA followed by the Dunnet's posttest was used. A p value of less than 0.05 was considered to be statistically significant. The results were obtained from groups of at least five experiments with different tissues from distinct animals.
2.3. Serum testosterone determination At the end of treatment, blood samples were obtained from control and treated animals. Then, serum was obtained by centrifugation and frozen until the moment of hormone determination. Serum testosterone levels were determined by chemiluminescence immunoassay (Access Testosterone kit, Beckman Coulter), with a sensitivity of 10 ng/dl. The analysis was performed at DNA Center Laboratory (Natal, Brazil).
2.7. Drugs and reagents Phenylephrine, carbachol, fluoxetine (for in vitro experiments) and sertraline (for in vitro experiments) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Fluoxetine and Sertraline used for the in vivo treatments were from Viafarma (Brazil) Iberoquímica Farmacêutica (Brazil), respectively. All reagents used for physiological salt solution were from Dinâmica (Brazil). 4
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Fig. 4. Effects of in vivo treatment with drug-free vehicle (control), fluoxetine or sertraline 20 mg/kg for 21 days on spontaneous contractions of rat distal cauda epididymis. Panel A - Original tracings showing the spontaneous contractions of distal cauda epididymis from control, fluoxetine or sertraline treated animals. Panel B and C – Bar graphs showing the effects of in vivo treatment with drugfree vehicle (control), fluoxetine or sertraline 20 mg/kg for 21 days on frequency (Panel B) and amplitude (Panel C) of spontaneous contractions of rat distal cauda epididymis. Each bar represents the means ± S.E.M. of 8–10 independent experiments performed with tissues from different rats. *p < 0.05 in relation to the control.
Fig. 5. Effects of in vivo treatment with drug-free vehicle (control), fluoxetine or sertraline 20 mg/kg for 21 days on rat distal cauda epididymis contractions induced by KCl (Panel A), phenylphrine (Panel B) or carbachol (Panel C). Each point represents Means ± S.E.M. of 8–10 independent experiments performed with tissues from different rats.
3. Results
decreased the KCl-induced distal cauda epididymis contraction by about 30 and 70%, respectively (Fig. 1A). The pre-incubation of fluoxetine 3 and 10 μM were also able to significantly diminish the Emax for phenylephrine (~50 and ~80%, respectively) (Fig. 1B; Table 1) or carbachol (~50 and ~75%, respectively) (Fig. 1C; Table 1). Thus, we also found that phenylephrine was 3-fold more potent in tissues from rats pre-exposed to fluoxetine 1 μM (Fig. 1B; Table 1). On the other hand, phenylephrine and carbachol were 5.5-fold and 7.5-fold,
3.1. In vitro effects of fluoxetine or sertraline on rat distal cauda epididymis contractions The Fig. 1 shows that fluoxetine or sertraline at concentrations higher that 3 μM induced a non-specific reduction in the contractile effects of KCl, phenylephrine or carbachol. Fluoxetine 3 and 10 μM 5
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0.31 ± 0.15, n = 10; Fluoxetine: 2.27 ± 0.48, n = 8; P < 0.05) and amplitude (Control: 0.026 ± 0.015, n = 10; Fluoxetine: 0.15 ± 0.02, n = 8; P < 0.05) of spontaneous contractions of rat distal cauda epididymis while sertraline treatment increased the amplitude (Control: 0.026 ± 0.015, n = 10; Sertraline: 0.11 ± 0.02, n = 8; P < 0.05) without affecting the frequency (Control: 0.31 ± 0.15, n = 10; Sertraline: 0.95 ± 0.28, n = 8; P > 0.05) (Fig. 4). The effects of fluoxetine or sertraline in vivo treatment were also checked on the contractions of rat distal cauda epididymis induced by the depolarizing agent KCl, phenylephrine (agonist of α1-adrenoceptors) or carbachol (agonist of muscarinic receptors) (Fig. 5). The KClinduced distal cauda epididymis contractions were not altered by the in vivo treatment with fluoxetine of sertraline (Fig. 5A). On the other hand, phenylephrine-induced contractions were 3- and 2.3-fold more potent in distal cauda epididymis from fluoxetine and sertraline-treated animals, respectively (Fig. 5B; Table 5). The potency for carbachol was unchanged after treatment with both fluoxetine or sertraline (Fig. 5C, Table 5). Furthermore, no significant changes were found in the maximum effects (Emax) for phenylephrine or carbachol in the treated groups when compared to control (Fig. 5C; Table 5).
Table 5 Maximal contractions (Emax) (% KCl contraction) and pEC50 values for phenylephrine or carbachol in rat distal cauda epididymis from rats submitted to the in vivo treatment with drug-free vehicle (control), fluoxetine or sertraline 20 mg/kg for 21 days. Pharmacological parameters Phenylefrine
Emax
pEC50
Control (n = 10) Fluoxetine 20 mg/kg (n = 8) Sertraline 20 mg/kg (n = 9) Carbachol Control (n = 10) Fluoxetine 20 mg/kg (n = 8) Sertraline 20 mg/kg (n = 10)
0.63 ± 0.03 0.60 ± 0.04 0.74 ± 0.05
6.16 ± 0.09 6.60 ± 0.08a 6.46 ± 0.05a
0.57 ± 0.03 0.55 ± 0.05 0.70 ± 0.05
5.81 ± 0.07 6.05 ± 0.08 5.75 ± 0.06
Emax (maximal contraction) expressed in g of tension and pEC50 (potency, measured as the negative log of EC50) obtained from the non-linear regression curves shown in Fig. 5B and C. Values are Means ± S.E.M. n, number of experiments. aP < 0.05 in relation to control.
respectively, less potent in the presence of fluoxetine 10 μM (Fig. 1B; Table 1). Sertraline 3 and 10 μM also decreased the contractions of rat distal cauda epididymis induced by a single concentration of KCl by about 40 and 75%, respectively (Fig. 1D). The pre-incubation of sertraline 3 and 10 μM depressed the Emax for phenylephrine (~35 and ~70%, respectively) (Fig. 1E; Table 2) or carbachol (~50 and ~80%, respectively) (Fig. 1F; Table 2). The phenylephrine and carbachol potency was decreased by 3- and 5-fold, respectively, after pre-incubation of sertraline 10 μM (Fig. 1E and F; Table 2). Sertraline 1 μM did not induce any alterations in the contractions of rat distal cauda epididymis induced by KCl, phenylephrine or carbachol (Fig. 1; Table 2).
4. Discussion This study showed that in vitro fluoxetine or sertraline (> 3 μM) impaired the contractions induced by KCl or exogenous agonists although fluoxetine 1 μM potentiate the phenylephrine-induced rat distal cauda epididymis contractions. Furthermore, in vivo treatment with fluoxetine and sertraline decreased daily sperm production, sperm reserves trough epididymis and sperm transit time in cauda epididymis in the rats. Thus, these aforementioned in vivo effects found in treated animals could be attributed to the reduction of serum testosterone levels, the enhancement of spontaneous contractions or potentiation of adrenergic agonist-induced distal cauda epididymis contractions. The motor activity of smooth muscle cells from epididymal duct is an important process to the transport of spermatozoa within the epididymis. It is widely accepted that during these transit trough epididymis, spermatozoa undergo maturation, acquire motility and fertilizing capacity (Dacheux and Dacheux, 2014; Elfgen et al., 2018). Several mechanisms may be important to the regulation of smooth muscle function in epididymis such as neuronal input (adrenergic and cholinergic input), hormonal, epithelial and sperm or luminal factors (Elfgen et al., 2018). Thus, any drug that interferes in smooth muscle contractility or its regulation process might affect the epididymis contraction, preventing the correct maturation and transport of sperm (da Silva Junior et al., 2014; Elfgen et al., 2018). It has been reported that both fluoxetine and sertraline negatively affect the smooth muscle contraction. For instance, fluoxetine or sertraline > 3 μM reduced the contractions induced by KCl or adrenergic agonists in rodent (Busch et al., 2000; Kalyoncu et al., 1999; Pedroso et al., 2017) or human vas deferens (Medina et al., 2000). Sertraline also inhibited the acetylcholine-induced guinea pig bladder contractions (Uno et al., 2017) and relaxed the pre-contracted human isolated mesenteric arteries (Vila et al., 1999). Therefore, we hypothesized that both fluoxetine and sertraline could also affect rat epididymis contraction. In our study, both fluoxetine and sertraline (> 3 μM) presented an inhibitory effects on KCl, phenylephrine or carbachol induced contractions, as characterized by a significant decrease in the maximum effect. This inhibitory effect could be attributed to the blockade of calcium channels, as it has been argued by previous studies (Busch et al., 2000; Kalyoncu et al., 1999; Medina et al., 2000; Pedroso et al., 2017; Ungvari et al., 2000; Uno et al., 2017). In fact, Deak et al. (2000) using patch-clamp technique in the whole-cell configuration reported voltage-gated calcium channels were inhibited by fluoxetine. Similarly, it is also described that sertraline presented the same pharmacological behavior (Lee et al., 2012). This antagonistic effect of fluoxetine and
3.2. Effects of chronic treatment with fluoxetine or sertraline on animals and reproductive organ weight Animals treated with drug-free vehicle presented a weight gain of 73.57 g ± 5.17 (n = 12) after 21 days of the treatment while fluoxetine and sertraline-treated rats showed a significant weight loss (P < 0.05 compared to control) of −54.45 g ± 6.56 (n = 11) and −38.8 g ± 13.41 (n = 12), respectively (Fig. 2). The in vivo treatment with fluoxetine and sertraline for 21 days also reduced the vas deferens, empty vesicle seminal, testis and epididymis weight when compared to control (Table 3). 3.3. Effects of chronic treatment with fluoxetine or sertraline on rat sperm production, sperm reserves, sperm transit time trough epididymis and testosterone levels The in vivo treatment with fluoxetine or sertraline for 21 days decreased the number of spermatids in the rat testis and the daily sperm production (DSP) as well (Table 4). A significant decrease was also found in the sperm reserves in caput/corpus and cauda of epididymis from fluoxetine and sertraline-treated animals. Furthermore, the treatment with fluoxetine or sertraline accelerated the sperm transit time trough rat cauda epididymis while sperm transit time of caput/body epididymis was unaltered (Table 4). We also found a decrease in serum testosterone levels in fluoxetine or sertraline-treated animals by about 60 and 55%, respectively, when compared to control (Fig. 3). 3.4. Effect of chronic treatment with fluoxetine or sertraline on rat distal cauda epididymis contractions The Fig. 4 shows the effects of in vivo treatment with fluoxetine or sertraline on spontaneous contractions of rat distal cauda epididymis. Fluoxetine treatment significantly augmented the frequency (Control: 6
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fluoxetine and sertraline accelerated the sperm transit time in rat cauda epididymis, plausibly indicating once more an alteration in motor activity of this epididymal site. In other to check this hypothesis, we assessed the spontaneous and drug-induced rat distal cauda epididymis contractions by functional experiments. Isolated distal cauda epididymis showed an increase in the spontaneous motor activity in treated animals, characterized by an increase in the frequency and/or amplitude of these contractions. The autorhythmicity of epididymal duct contractions are important to provide spermatozoa propulsion through the epididymal duct, ensuring sperm maturation and male fertility (Elfgen et al., 2018; Mewe et al., 2006). This process seems to be originated from epithelial PGF (2alpha)-release (Mewe et al., 2006) and it is under control of several factors, including testosterone (Elfgen et al., 2018). Actually, studies indicated that testosterone has important regulatory effects on smooth muscle contractility of genitourinary tract, as demonstrated by the development of spontaneous contractions in rodent vas deferens or seminal vesicle after castration (Hib et al., 1985; MacDonald and McGrath, 1980), increasing of spontaneous contraction amplitudes in caput/ corpus, and augmented basal intraluminal pressure in corpus and cauda epididymis after castration (Din-Udom et al., 1985). Therefore, we suggest that fluoxetine and sertraline treatment increased the spontaneous contractions, and consequently, the sperm transit time of rat distal cauda epididymis indirectly by diminishing the serum testosterone levels. Epididymal smooth muscle contractions elicited by adrenergic and cholinergic neurotransmitters via α1-adrenoceptors and M3 receptors, respectively, also play a significant role on sperm transport trough epididymis, mainly in epididymal cauda (Dacheux and Dacheux, 2014; Elfgen et al., 2018). Then, we also checked the effects of long-term in vivo treatment with fluoxetine or sertraline on rat distal cauda epididymis contractions induced by phenylephrine (α1-adrenoceptors) or carbachol (muscarinic receptor agonist). Phenylephrine was more potent to induce epididymal contractions in fluoxetine and sertraline treated animals compared with controls, while no significant alterations were found for carbachol or KCl-induced contractions. A potentiation of noradrenaline contractile responses was previously observed in vas deferens from animals chronically treated with fluoxetine (Busch et al., 1999) or sertraline (Ozyavuz et al., 2004). The mechanism underlying the aforementioned effects of fluoxetine or sertraline is not fully understood, but it was proposed that both drugs could induce supersensitivity due to noradrenaline exhaustion caused by prolonged inhibition of noradrenaline uptake system. More studies are needed for a comprehensive view of fluoxetine and sertraline longterm effects on adrenergic neurotransmission of genitourinary tract. However, the potentiation of adrenergic-induced contractions by both drugs could be another important mechanism to accelerate the sperm transit trough rat cauda epididymis.
sertraline on calcium channels seems to play a major role in its inhibitory actions on smooth muscle contraction. Fluoxetine 1 μM potentiated the phenylephrine-induced contractions of rat distal cauda epididymis without affecting the carbachol potency, indicating a selective action on epididymal adrenergic system. Similar observation was reported by Busch et al. (2000) who showed that in vitro fluoxetine increased the contractile effects of low noradrenaline concentrations in rat vas deferens, and such effect was attributed to a putative blockade of noradrenaline reuptake process. Although considered a selective serotonin reuptake inhibitor, it is reported that fluoxetine at high concentrations also inhibits the noradrenaline transporter (estimated KD 1.56 μM) (Wenthur et al., 2014). Considering that phenylephrine is also susceptible to neuronal noradrenaline transporter (8.5-fold less than noradrenaline) (Iversen, 1967), we postulate that the leftward shift of phenylphrine curves in the presence of fluoxetine could be associated to its interference on noradrenaline neuronal uptake system in rat epididymis. Conversely, fluoxetine 10 μM decreased the potency of phenylephrine and carbachol. The rightward shift of phenylephrine or carbachol curves in the presence of fluoxetine could be consequence of its affinity for both α1adrenoceptors (estimated KD 2.26 μM) and M3 receptors (estimated KD 1 μM) (Wenthur et al., 2014). Sertraline 10 μM showed the same pharmacological behavior than fluoxetine 10 μM on the potencies for phenylephrine and carbachol. It is described that sertraline is able to inhibit the α1-adrenoceptors (estimated Ki 36–190 nM) and muscarinic receptors (estimated Ki 200 nM) (Owens et al., 1997; Sanchez et al., 2014). Then, the off-targets effects of sertraline on α1-adrenoceptors and muscarinic receptors could be associated to the reduction of phenylephrine or carbachol potency in the presence of this drug. Sertraline is also able to inhibit the noradrenaline neuronal uptake system (estimated Ki 420–820 nM) (Sanchez et al., 2014), but no potentiation of phenylephrine responses was found in our study. This could be probably due to the relative high affinity of sertraline for α1-adrenoceptors that could counteract its effects on noradrenaline uptake system. It is also noteworthy to cite that the negative contractile effects of both fluoxetine or sertraline (on Emax or potency) in rat epididymis was often found in concentrations higher than 3 μM, and therefore, it is still debatable the therapeutic implication of this off-target effects. We found that long-term in vivo treatment of rats with fluoxetine or sertraline induced a marked decrease in daily sperm production and serum testosterone levels. These results are similar to those reported earlier which showed that both fluoxetine and sertraline decrease androgen production in rodents or cell lines (Camara et al., 2019; Hansen et al., 2017; Jahromy and Moghadam, 2014; Munkboel et al., 2018), leading to a spermatogenesis impairment (Aggarwal et al., 2012; Bataineh and Daradka, 2007; Camara et al., 2019; Erdemir et al., 2014). The exact mechanism underlying the negative effects of fluoxetine or sertraline on testicular steroidogenesis is still under investigation, but recent evidence suggest these drugs are able to disrupt androgen synthesis by interfering on steroidogenic enzymes activity (Hansen et al., 2017; Munkboel et al., 2018). Moreover, the reduction in testosterone levels could also be associated in the decrease of animal and organ weights, although other mechanisms not explored for this study could be involved (Scabia et al., 2018; Silverstein-Metzler et al., 2016). Chronic treatment with fluoxetine or sertraline are able to reduce sperm counts in human ejaculate (Akasheh et al., 2014; Safarinejad, 2008) or rodent cauda epididymis (Atli et al., 2017; Bataineh and Daradka, 2007). Likewise, we also found a decrease in sperm number in rat epididymis after treatment with both SSRIs. This may be a direct consequence of testosterone decreased production or alterations in sperm transport along epididymis, since drugs (i.e. sibutramine, bisphenol-A, diethylstilbestrol, indomethacin etc) that are known to affect steroidogenesis and/or epididymis contractions lead to a sperm count reduction (Bagoji et al., 2017; Bellentani et al., 2011; Cariati et al., 2019; Fernandez et al., 2008). Further, we also found that
5. Conclusion In conclusion, it is clear that in vitro or long-term treatment with fluoxetine and sertraline are able to affect rat epididymal contraction. The alteration in the motor activity of rat epididymis induced by fluoxetine and sertraline could be associated to the low sperm count in this organ and accelerated transit time trough epididymal cauda. Altogether, our data indicate that epididymis might be an important target for the anti-fertility effects of fluoxetine or sertraline. Declaration of competing interest The authors declare no conflict of interest. Acknowledgments Thanks are due to Flávio Maurílio dos Santos Lima, Cesar Augusto 7
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Trevisan Bordignon and Ramon Tadeu Galvão Alves Rodrigues for excellent technical support and to the Pro-Rectory of Research of Federal University of Rio Grande do Norte (Portuguese: Pro-Reitoria de Pesquisa da Universidade Federal do Rio Grande do Norte), CNPq and CAPES for financial support.
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