Menoufia Medical Journal

ORIGINAL ARTICLE
Year
: 2017  |  Volume : 30  |  Issue : 4  |  Page : 1135--1142

Effect of antidepressant drug (fluoxetine) on the testes of adult male albino rats and the possible protective role of omega-3


Maha E Soliman, Bothina L Mahmoud, Maisa A Kefafy, Rania I Yassien, Eman S. A. El-Roghy 
 Department of Histology, Faculty of Medicine, Menoufyia University, Sheibin El Kom, Egypt

Correspondence Address:
Eman S. A. El-Roghy
Department of Histology, Faculty of Medicine, Menoufyia University, Sheibin El Kom, Menoufia
Egypt

Abstract

Objective The aim of this study was to evaluate the possible protective effect of omega-3 on testicular tissue damage induced by antidepressant drug (fluoxetine), in adult male albino rats. Background Fluoxetine is a selective serotonin reuptake inhibitor drug commonly used in the treatment of depression, but it causes structural changes of testicular tissue. Omega-3 has a range of anti-inflammation and antioxidation. Materials and methods This animal study was carried out during the period from December 2016 to June 2017 on 50 adult male albino rats that were kept in the animal house of the Faculty of Medicine, Menoufiya University, and divided into four groups: group I (control); Group II (omega-3); group III, subdivided into two subgroups – IIIa (fluoxetine) and IIIb (recovery); and group IV (fluoxetine and omega-3). All groups received drugs orally for 4 weeks, and the recovery subgroup was left untreated for another 4 weeks. Then, animals were sacrified and weighted. Testis samples were obtained for histological and immunohistochemical study. Results Fluoxetine-treated rats showed a highly significant decrease of body and testis weight (P < 0.001). Fluoxetine led to distortion of seminiferous tubules, germ cell degeneration with sloughing, and vacuolation. The interstitium appeared wide containing degenerated Leydig cells, congested blood vessels, and acidophilic material. Fluoxetine induced intense expression of caspase-3, decrease in the mean number of positive proliferating cell nuclear antigen immunostaining, and negative expression of androgen receptor. The recovery group still showed persistence of some changes. Fluoxetine and omega-3 groups showed marked improvement. Conclusion Omega-3 alleviates testicular damage induced by fluoxetine more than arresting fluoxetine.



How to cite this article:
Soliman ME, Mahmoud BL, Kefafy MA, Yassien RI, El-Roghy ES. Effect of antidepressant drug (fluoxetine) on the testes of adult male albino rats and the possible protective role of omega-3.Menoufia Med J 2017;30:1135-1142


How to cite this URL:
Soliman ME, Mahmoud BL, Kefafy MA, Yassien RI, El-Roghy ES. Effect of antidepressant drug (fluoxetine) on the testes of adult male albino rats and the possible protective role of omega-3. Menoufia Med J [serial online] 2017 [cited 2021 Oct 28 ];30:1135-1142
Available from: http://www.mmj.eg.net/text.asp?2017/30/4/1135/229228


Full Text



 Introduction



Depression is a common chronic recurrent mood disorder that influences both economic and social functions worldwide. Depression is observed more frequently at reproductive ages[1]. Infertility and problems related to it are one of the most vital issues in the life of couples. By statistics, male infertility represents about 35% of infertilities. Drugs have been reported to play a role in the etiology of male infertility. Fluoxetine is one of the selective serotonin reuptake inhibitor drugs (SSRI) used in neurological disorder treatment such as depression, bulimia nervosa, and obsessive-compulsive disorder[2]. The effect of fluoxetine on male fertility was studied by some authors. Administration of fluoxetine caused a decrease in spermatogenesis and weights of reproductive organs[3]. Sexual disorders and decreased semen parameters were reported in patients treated with antidepressant fluoxetine[4].

Omega-3 is a polyunsaturated fatty acid that cannot be synthesized in vivo, so it is considered as one of the essential polyunsaturated fatty acids. It is derived from fish oil, flaxseed, and some nuts[5]. Jabbar and Mahde[6] proved that omega-3 has powerful protective effect on male reproductive system of rats that were exposed to toxic substance. In addition, Kumar et al.[7] showed that omega-3 has anti-inflammatory and antioxidant effects protecting against testicular toxicity. Furthermore, Olutope et al.[8] studied the protective effect of omega-3 on testicular lipid concentration in rats.

This work was designed to evaluate the possible protective effect of omega-3 on testicular tissue damage induced by antidepressant drug (fluoxetine), in adult male albino rats, and the possible recovery after cessation of treatment.

 Materials and Methods



Materials

Animals

This animal study was carried out during the period from December 2016 to June 2017 on 50 adult male albino rats weighing 190–200 g. Animals were housed in clean properly ventilated cages, fed on a standard laboratory diet, and maintained on a 12-h light/dark photoperiod in the animal house of the Faculty of Medicine, Menoufiya University, Sheibin El Kom, Menoufia, Egypt. The animals were treated in accordance with the guidelines approved by the Animal Care and Ethical Committee of Faculty of Medicine, Menoufiya University.

Animals were randomly divided into four groups:

Group I (control) comprised 10 rats; half of them received corn oil 'vehicle of omega 3', and the other half received distilled water 'vehicle of fluoxetine' orally by gavage once daily for 4 weeks.

Group II (omega-3 treated) comprised 10 rats that were administered 400 mg/kg/day of omega-3 dissolved in corn oil orally by gavage once daily for 4 weeks[5].

Group III comprised 20 rats that were further subdivided into two equal subgroups:

Subgroup IIIa (fluoxetine treated) received fluoxetine (10 mg/kg/day) dissolved in distilled water orally by gavage once daily for 4 weeks. This dose is equivalent to the therapeutic dose for humans[2].

Subgroup IIIb (recovery) was administered fluoxetine orally for 4 weeks as in subgroup IIIa, and then left untreated for a further 4 weeks.

Group IV (fluoxetine and omega-3 treated) comprised 10 rats that received a combination of fluoxetine (10 mg/kg/day) and omega-3 (400 mg/kg/day) orally by gavage once daily for 4 weeks.

The total time of experiment is 8 weeks: 4 weeks for treatment and another 4 weeks for recovery.

Drugs

Fluoxetine (Philozac) capsules each contained 10 mg, which is manufactured by Amoun Pharmaceutical Company, Cairo, Egypt.

Omega-3 (Super omega) was obtained as gelatin capsules each containing 1000 mg from Safe Pharmaceutical Company Cairo, Egypt for Regal pharma under license of Majestic Power Co., USA, Egypt.

Methods

All animals were weighed before the experiment and daily cage-side examinations were performed to examine rat behavior. At the end of the experiment, animals were sacrified and weighed. The testes of all groups were dissected out and their weights were recorded. For histological examination, the testes were fixed in Bouin's solution and processed for paraffin blocks. Sections of 4 μm were cut and stained with hematoxylin and eosin (H and E), Masson Trichome (MT), and Periodic Acid Schiff's reaction (PAS) stain[9].

Immunohistochemical staining was performed on 4-mm-thick sections. Endogenous peroxidase activity was blocked with 0.3% hydrogen peroxide. For antigen retrieval, citrate buffer was used after the sections were treated in a microwave. The tissue sections were incubated overnight at room temperature with the following primary antibodies: anti-caspase-3 [mouse monoclonal antibody (Lab Vision, Fremont, CA USA)[10], antiprimary antibodies to proliferating cell nuclear antigen (PCNA) (Clone PC 10; Dako Denmark A/S, Glostrup, Denmark)[11], and primary monoclonal and polyclonal antibodies for androgen receptors (Cat. No. MA1-150; Thermo Fisher Scientific Co., Waltham, Massachusetts, USA)[12]. The staining was completed using the streptavidin–biotin complex detection method. Controls slides were included in each run. Negative controls were prepared by excluding the primary antibody and replacing it with PBS.

Morphometric study

Five images were captured from randomly selected PCNA sections per group. Images were taken at ×200 magnification. The number of immune-positive cells in each seminiferous tubule was counted using image J software Open source, contributors worldwide [Figure 1]a. Only the basal germ cells of these tubules were counted, because they are the cells where active DNA synthesis took place[12]. The calculated numbers were considered for comparison and statistical analyses.{Figure 1}

Statistical analysis

Data were statistically analyzed by SPSS, version 20 (SPSS Inc., Chicago, Illinois, USA). Data were expressed as mean ± SD and analyzed by using one-way analysis of variance test followed by post-hoc test for comparison between all groups. Differences were regarded as significant if P values were less than 0.05.

 Results



Histological and immunohistochemical results

Group I (control)

Examination of H and E-stained sections from control animals showed that the testicular parenchyma was formed of densely packed seminiferous tubules with rounded, regular outline, and stratified germinal lining. These tubules were enclosed by a connective tissue capsule (tunica albuginea) [Figure 1]b. The germinal epithelium was formed of spermatogonia, primary spermatocytes, spermatids, and sertoli cells. Primary spermatocytes were the largest ones seen within the seminiferous tubules, and some showed mitotic figures. Spermatids were small cells, rounded or elongated, arranged in 2–4 rows lying close to the lumen of the tubules. Sertoli cells were detected on the basement membrane between the spermatogenic cells. They were columnar cells with irregular outlines. Their nuclei tend to be triangular. The myoid cells were arranged outside the basal lamina [Figure 1]c. The interstitium contained clusters of interstitial Leydig cells with acidophilic cytoplasm [Figure 1]d. Normal distribution of minimal collagen fibers in the testicular capsule, basal lamina, and interstitial tissues were detected by MT stain [Figure 1]e. PAS-stained section showed moderate reaction in basal lamina of tubules [Figure 1]f.

Immunohistochemical stained sections of the control group revealed negative caspase-3 immunoexpression [Figure 2]a, positive PCNA immunostaining (deep brown nuclear reaction) in all nuclei of basal germ cells [Figure 2]b, and positive androgen receptor immunoexpression. It appeared as brown nuclear staining of the interstitial cells, myoid cell, and some sertoli cells [Figure 2]c.{Figure 2}

Group II (omega-3 treated)

Sections of this group showed normal histological features nearly similar to the control group. The seminiferous tubules contained different germ cells and several mature spermatozoa appeared in the lumen. Normal Leydig cells were seen in-between the tubules [Figure 2]d. Minimal collagen fiber deposition and moderate PAS reaction were noticed. Immunohistochemical stained sections of this group were similar to those of control [Figure 2]e and [Figure 2]f.

Subgroup IIIa (fluoxetine treated)

Fluoxetine induced variable degrees of tubular affection in the form of distorted seminiferous tubules, cellular disorganization, germ cells sloughing, and cytoplasmic vacuolation. The tubules were surrounded by thick irregular basal lamina. The lumen of tubules contained few spermatozoa. Some germ cells had deeply stained pyknotic nuclei. Acidophilic hyaline streaks were extended between the degenerated spermatogenic cells. The intertubular space appears wide containing degenerated distorted Leydig cells and congested dilated interstitial blood vessels. Acidophilic hyaline material and vacuolations were present in most of the interstitial spaces [Figure 3]a,[Figure 3]b,[Figure 3]c,[Figure 3]d,[Figure 3]e. There was a marked increase of the collagen fiber deposition in the capsule, basal lamina, and interstitium [Figure 3]f. Very strong PAS reaction in the basal lamina, intertubular space, and around blood vessels was detected [Figure 3]g. Immunohistochemically, fluoxetine treatment induced a marked increase in cytoplasmic expression of caspase-3, decrease in the mean number of PCNA-positive immunostaining germ cells, and negative immunoexpression of androgen receptor [Figure 3]h,[Figure 3]i,[Figure 3]j.{Figure 3}

Subgroup IIIb (recovery)

The testis of the recovery group still contained distorted seminiferous tubules that did not return to normal. Acidophilic material and vacuolations were present in the interstitium [Figure 4]a and [Figure 4]b. Massive collagen fibers were noticed in the capsule, around blood vessels, interstitial tissues, and in the basal lamina [Figure 4]c. There was strong PAS reaction in basal lamina and around blood vessels [Figure 4]d. Immunohistochemically, this group still showed few cells with intense positive cytoplasmic caspase-3 immunostaining, positive PCNA immunostaining in some germ cells, and weak androgen receptor immunostaining [Figure 4]e,[Figure 4]f,[Figure 4]g.{Figure 4}

Group IV (fluoxetine and omega-3 treated)

Marked improvement was observed in the examination of testes of this group. Most tubules were more or less similar to those of control, and they were lined by normal arrangement of spermatogenic cells. But some cells had vacuolated cytoplasm. The intertubular space remained wide and contained acidophilic material [Figure 5]a and [Figure 5]b. Moderate collagen fiber deposition [Figure 5]c and moderate PAS reactions were detected [Figure 5]d. This group showed improved immunohistological results nearly similar to control [Figure 5]e,[Figure 5]f,[Figure 5].{Figure 5}

 Statistical Results



The animal body and testis weight

The mean of both body and testis weight of omega-3-treated group II showed nonsignificant relation compared with the control group (P = 0.082 vs. 0.86, respectively). The mean body and testis weight of the fluoxetine-treated subgroup IIIa showed a highly significant decrease (P< 0.001), whereas that of the recovery subgroup IIIb showed a significant decrease compared with control (P = 0.012 vs. 0.001, respectively). However, fluoxetine and omega-3-treated group IV showed a nonsignificant change (P = 0.129 and 0.48, respectively) [Table 1].{Table 1}

The number of positive proliferating cell nuclear antigen cells

The mean number of positive PCNA cells of omega-3-treated group II showed a nonsignificant change as compared with the control group (P = 0.218). The mean number of positive PCNA cells of fluoxetine-treated subgroup IIIa showed a highly significant decrease (P< 0.001), whereas that of recovery subgroup IIIb showed a significant decrease (P 0.01) compared with control. However, fluoxetine-treated and omega-3-treated group IV showed a nonsignificant change compared with control (P = 0.099) [Table 1].

 Discussion



Fluoxetine is a strong selective serotonin reuptake inhibitor widely prescribed for the treatment of neurological disorders such as depression and anxiety[3].

This work was designed to evaluate the possible protective effect of omega-3 on testicular tissue damage induced by fluoxetine and the possible recovery after cessation of treatment.

High significant reduction of body and testis weight in fluoxetine-treated rats was noticed in the present study. These results were in harmony with those of other investigators such as El-Kholey and Abdel-Salam[13], Sakr et al.[3], and Hajizadeh et al.[14]. The latter stated that fluoxetine has inhibitory actions on the gastrointestinal tract and decreases the height of intestinal villus leading to decrease the intestinal absorption and reduction of necessary nutrients. As mentioned by Unnikrishnan et al.[15], treatment of animals with fluoxetine caused hypoglycemia and a decrease in both testicular protein and cholesterol, leading to a decline in the testis weight. According to Hajizadeh et al.[14], testicular weight largely depends on the population of differentiated spermatogenic cells. Therefore, significant testicular weight loss induced by fluoxetine can be attributed to testicular hypocellularity and spermatogenesis suppression. However, Ramos et al.[16] proved that fluoxetine treatment had no effect in body weight and testicular weight of male rat newborn after maternal exposure to fluoxetine.

Fluoxetine has injurious effects on the testicular tissue including distortion of the seminiferous tubules, degenerated spermatogenic cells with large cytoplasmic vacuoles, and sloughing of germ cells. These findings were previously reported by several number of researchers as Hajizadeh et al.[14] and Atli et al.[17].

Many authors explained the mechanism by which fluoxetine induces testicular tissues toxicity. Inkielewicz-Stępniak[18] showed that fluoxetine induces lipid peroxidation leading to free-radical release, which causes membrane disorganization and subsequent decreases in membrane fluidity, and finally extensive tissue damage. Recently, Atli et al.[17] proved that SSRIs induce DNA fragmentation and reactive oxygen species overproduction, as a result of oxidative stress, which induces cell damage in male rat's reproductive organs.

Cytoplasmic vacuolation and degeneration might be considered an indication of cell necrosis. According to Manivannan et al.[19], vacuolations of germ cells could be a result of metabolic disturbance in these cells with a subsequent change in their morphology.

Sloughing of germ cells was suggested to be due to the effect on the organization of germ cells that are held in place by a close relation between their membranes and specialized junctions of Sertoli cell membrane. The affected Sertoi cells induced changes or decreases in seminiferous tubule fluid secretion, which further resulted in apical sloughing and germ cell death[19].

Some germ cells had deeply stained pyknotic nuclei. Such finding was confirmed by Atli et al.[17], who considered pyknosis as a sign of germ cell degeneration.

Few sperm inside the lumen of some seminiferous tubules and were no sperm in other tubules were also noticed in this study. This was in agreement with the study by Kumar et al.[20], who proved that fluoxetine had a spermicidal effect. They also proposed that the SSRIs bound to sulfhydryl groups in the sperm plasmalemma and interfere with ATP synthesis in the sperm by interacting with phospholipids.

The presence of wide spaces between the tubules in this study suggests the presence of interstitial edema, as had been previously reported by El-Kholey and Abdel-Salam[13]. According to Ahmed and Shaheen[21], the intertubular homogeneous acidophilic material detected in this study could be attributed to excess lymphatic exudates oozing from degenerated lymphatic vessels or owing to an increase in vascular permeability. Dilated congested blood vessels were observed in some areas of dilated interstitium. This was in harmony with El-Kholey and Abdel-Salam[13] and could be attributed to a compensatory mechanism of decreased testicular blood flow following fluoxetine administration. Also, Aggarwal et al.[4] stated that fluoxetine induces interstitial tissue inflammation and fibrosis, leading to a decrease in the blood supply.

The interstitial cells of Leydig showed degeneration that could be explained as fluoxetine decreased blood supply led to atrophy of Leydig cells[4],[17].

Thickened connective tissue capsule (tunica albuginea) was observed in this study. It was reported that this thickening is accompanied by a decrease in the rate of sperm production[22].

The marked deposition of collagen fibers, in addition to the presence of strong PAS reaction, was attributed to the oxidative stress induced by fluoxetine. This explanation coincided with the finding of El-Din and Abd-El Aty[5], who reported that the formation of hydroxyl radical and other highly reactive oxidizing molecules led to lipid peroxidation causing damage to proteins and nucleic acids. The end results of these reactions led to an increase in collagen fibers and ground substance formation.

Fluoxetine increased expression of caspase-3 in spermatogenic cells and Leydig cells. Such finding coincided with those of Sakr et al.[3] and Atli et al.[17]. In addition, Khaksar et al.[23] stated that fluoxetine induces cytochrome-C release and caspase activation, leading to DNA fragmentation and germ cell apoptosis.

In the present study, fluoxetine-treated testis showed few positive PCNA immunostaining cells. These findings indicated DNA damage according to El-Din and Abd-El Aty[5], who supposed that the oxidative stress and reactive oxygen species overproduction induced by fluoxetine led to depletion of the active DNA contents in these dividing cells, which finally led to depletion of spermatozoa.

It is well documented that the optimum level of testosterone is necessary for ordinary spermatogenesis and germ cell structural development. In support of these facts, it seems that fluoxetine induces disruption of testosterone level leading to defective spermatogenesis[14]. The androgen action is mediated by a single receptor, which is almost exclusively nuclear[24]. In the present study, fluoxetine-treated testis revealed negative immunoexpression of androgen receptor, and this is in harmony with many investigators such as Jalili et al.[2] and Hajizadeh et al.[14].

After arrestof fluoxetine treatment, there were partial improvements. The testis of this group still contained distorted seminiferous tubules. This group still showed few cells with intense caspase-3 expression, positive PCNA immunostaining in some germ cells, and weak androgen receptor. These findings may be supported by the study of Csoka et al.[25], in which they showed that symptoms of sexual dysfunction persist after drug discontinuing. Persistence of some alterations after stopping fluoxetine treatment may indicate the need for more time for recovery or using appropriate protective drug.

In the present study, omega-3 with fluoxetine led to marked improvement. However, most tubules were more or less similar to that of control, and had less deposition of collagen fibers and less PAS-positive material deposition compared with control. In addition, a nonsignificant change of body and testis weight compared with control was reported. These improvements were attributed to the powerful antioxidant effects of the omega-3[8],[26]. The mechanism of protective role of omega-3 was explained by Maneesh et al.[27], who stated that omega-3 fatty acids have anti-inflammatory properties that were based on their ability to antagonize the activity of arachidonic acid, thereby reducing the production of inflammatory and chemotactic derivatives and suppressing cell-mediated immune responses. The antioxidant and anti-inflammatory effects of omega-3 are through removing of free radicals and inhibiting lipid peroxidation[7]. Omega-3 also stimulates spermatogenesis and increases the activity of Sertoli cells[28].

Omega-3 administration along with fluoxetine also improved immunohistochemical results, where there was mild caspase-3 immunostaining and many positive PCNA immunostaining. These were in harmony with those obtained by Elelaimy et al.[29], who proved that omega-3 showed a highly significant decrease in the percentage of DNA fragmentation.

Intense androgen receptor immunoexpression was noticed in fluoxetine and omega-3-treated group and this finding coincided with that of El-Din and Abd-El Aty[5].

 Conclusion



Fluoxetine treatment of rats caused significant testicular damage and germ cell apoptosis, which probably may affect reproductive functions. These structural changes could be alleviated by arresting fluoxetine treatment. However, combined treatment of fluoxetine with omega-3 was more superior in testicular protection owing to its antioxidant properties. Therefore, it is recommended to use omega-3 whenever antidepressant drug such as fluoxetine is used.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

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