1846.pdf - :: Al- Azhar Assiut Medical Journal ::

AAMJ, Vol. 10, N. 3, Sep, 2012, Suppl-1

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264

BIOCHEMICAL AND IMMUNOCYTOCHEMICAL STUDIES

OF THE TESTICULAR CHANGES AFTER TREATMENT

WITH DULOXETINE HYDROCHLORIDE AND THE

POSSIBLE PROTECTIVE EFFECTS OF OMEGA 3 IN ADULT

RAT MODEL OF DEPRESSION

Sahar Badr El- Din* and Olfat A. Abd-El Aty

**

Departments of Pharmacology* and Anatomy** Faculty of Medicine Al-Azhar

University (Girls).

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ABSTRACT

The antidepressant drugs are among those drugs which cause toxic effects

on much of organs especially male reproductive system. Duloxetine is one of the

potent reuptake inhibitor of serotonin and norepinephrine (SNRIs). It is used for

the treatment of many major depressive disorders. Omega-3 (N-3) fatty acids

are essential, polyunsaturated fatty acids (PUFAs). It is commonly used as a

powerful antioxidant. Aim of work, to demonstrate the biochemical and

immunocytochemical changes in the testes of adult rat treated with Duloxetine

after induction of depression and to evaluate the possible protective effect of

Omega 3. Material and Methods, Eighty male albino rats were divided into

four equal groups, Group I non stressed: received orally distilled water, Group

II stressed rats treated with (400 mg/kg/day) Omega-3, Group III stressed rats

treated with Duloxetine hydrochloride (30 mg/kg/day) orally and Group IV

stressed rats treated with Duloxetine and Omega-3 for two months. At the end of

the experimental period the testosterone, LH and FSH were evaluated. Markers

of oxidative stress of testis were also investigated. Moreover, histopathological

and immunocytochemical examination using PCNA staining of the testicular

tissue were carried by light microscopy. Also, in-vitro, evaluation of the

contractions induced by norepinephrine on rat vas deferens was carried in all

Sahar Badr El- Din and Olfat A. Abd-El Aty ـــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ

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groups. The results: showed that treatment with Duloxetine caused significant

decreased in the testosterone, LH and FSH levels (P˂0.001). Moreover GSH,

SOD and CAT levels reduced significantly (P˂0.001) with increased in MDA

concentration (P˂0.001). Also, marked signs of cellular degeneration and

necrosis with great depletion of germ cells and Sertoli cells of the affected

seminiferous tubules .The germ cells were disorganized, disrupted and it was

difficult to differentiate between them, whereas most of these cells had pyknotic

nuclei. There were no sperms observed. In addition there was significant

decrease of PCNA immunostaining cells (P˂0.001) which provide an evidence

of exposure of the testes to oxidative stress. Addition of Omega 3 to group IV

gave a statistically significant increase in the levels of testosterone, LH , FSH ,

GSH, SOD and CAT (P˂0.001) .Also, a statistically significant decrease in

MDA levels were determined when compared to Duloxetine treated group. In

addition, moderate regeneration and improvement were observed in the

histopathological examination whereas; some seminiferous tubules contain

many rows of different stages of spermatogenic cells and Sertoi cells in-

between. Small amount of mature spermatozoa appeared in the lumina.

Significant increases (P˂0.001) of PCNA immunostaining cells were present

when compared with groups III. In-vitro, Duloxetine significantly attenuate the

contractions induced by norepinephrine on rat vas deferens. Conclusion:

Duloxetine administration induced oxidative stress leading to quantitative and

qualitative alterations in the hormonal levels, oxidative parameters, process of

spermatogenesis and the contractility of vas deferens. Omega-3, preserve

adequate testicular functions against disturbances caused by Duloxetine.

Keywords: Duloxetine, Omega-3, Testes, Vas deferens, Oxidative stress,

Histopathology, Immunohistochemical, PCNA.

Sahar Badr El- Din and Olfat A. Abd-El Aty


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INTRODUCTION

The antidepressants drugs are among those drugs which cause toxic effects

on much of organs especially male reproductive system. About 15% of these

drugs have adverse effects on hormonal levels. It attacked target organs like

testes, which secrete hormones and produces male germ cells during

spermatogenesis. Studies showed that the effects of antidepressant on sexual

dysfunction are more than 60% (Williams et al., 2010). Duloxetine is one of the

newer potent reuptake inhibitor of serotonin and nor epinephrine (SNRIs). Its

chemical designation is [(+)-(S)-N-methyl-gamma- (1-naphthyloxy)-2-

thiophenepropylamine hydrochloride] (Kelly et al., 2002). It is effective for the

treatment of major depressive disorder (Dell'Osso et al., 2010), diabetic

neuropathic pain, stress urinary incontinence, generalized anxiety disorder and

fibromyalgia (Khan and Macaluso, 2009). Recent studies have also observed

that Duloxetine represents an effective switch strategy for the treatment of

SSRI-resistant major depression (Boyle et al., 2012).

Effects of drugs on spermatogenesis appear to be due to changes in hormones

level such as testosterone, LH, FSH and prolactin (Soghra et al., 2008). The

gonads and adrenals secrete several male sex hormones, androgens. All are

steroid hormones that derived from cholesterol. Testosterone is the most potent

and abundant androgen. Gonadotropin-releasing hormone (GnRH) secreted

from the hypothalamus promotes anterior pituitary release of luteinizing

hormone (LH) and follicle stimulating hormone (FSH). LH stimulates the

interstitial cells of Leydig in the testes to synthesize and secrete testosterone

(Freeman et al., 2001). FSH, which binds with specific FSH receptors attached

to the Sertoli cells in the seminiferous tubules, causing these cells to grow and

secrete various spermatogenic substances like nutrients, minerals and growth

factors required for the normal development of germ cells (Neill and Herbison,

2006).



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In the recent past, many pharmacological researches have been focused on

the effects of antioxidants in different pathological conditions (Victor et al.,

2004 ;Khan et al., 2010 and Obianime et al., 2010). The findings of such

studies have revealed the involvement of free radicals in most disease

conditions. Oxidative stress is linked to the pathogenesis of cardiovascular

dysfunction, e.g., hypertension, cerebrovascular accidents, and heart failure

(Aviram, 2000); cancer (Klaunig and Kamendulis, 2004); reproductive

dysfunction (Allen et al., 2004 and Santos et al., 2006); aging (Rattan, 2006)

neurodegenerative diseases (Nunomura et al., 2006) many age-related chronic

diseases, including atherosclerosis, diabetes mellitus (Szabo, 2009) and septic

shock (Anas et al., 2010).

Omega-3 (N-3) fatty acids are essential, polyunsaturated fatty acids

(PUFAs), i.e. they cannot be synthesized in vivo. In diet, large quantities are

found naturally in fish oil, flaxseed and some nuts. They derive from α-linolenic

acid and mainly occur as eicosapentaenoic acid (EPA) and docosahexaenoic

acid (DHA), which are both anti-inflammatory (Stulnig, 2003). These are then

converted to active metabolites, in particular, molecules known as resolvins and

protectins. These recently discovered lipid products are yet to be fully

characterized, but are thought to mediate, at least in part, the anti-inflammatory

and antioxidant effects of omega-3 fatty acids (Serhan, 2005). Omega-3 fatty

acids are key regulators of peroxisome proliferator-activated receptor alpha

(PPARα), which upregulate several genes associated with fatty acid and lipid

metabolism that stimulate fatty acid oxidation (Mishra et al., 2004 and

Nagasawa et al., 2006). Interestingly, there may be an independent, anti-

inflammatory and antioxidant effects via PPARα-mediated suppression of TNF-

α and IL-6 (Brown and Plutzky, 2007). The exact effects of Duloxetine on the

structures of the rat testes remain uncertain. Therefore, this experimental study

was designed to demonstrate the biochemical and immunocytochemical changes



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in the testes of adult rat after induction of depression and to evaluate the

possible protective effect of Omega 3.

MATERILES AND METHODS

A) Experimental animals:

Eighty male albino rats weight ranged between 250-300 gm. All animals

were housed with females (one male and two females ) at the animal house in

the Faculty of Medicine for Girls Al-Azhar University at 21°C–22°C in a 12

hr/12 hr light/dark cycle, fed standard rat chow, and given free access of water.

Rats were accommodated to the laboratory conditions one month before starting

the experiment. Male rats were divided into four equal groups.

Group I: Rats not received any stress were housed in a separate room and

received orally distilled water daily for 60 days

Group II: Rats model of chronic stress-induced depression received omega

3(400 mg/kg /day) orally for 60 days.

Group III: Rats model of chronic stress-induced depression received

therapeutic dose of Duloxetine (30 mg/kg /day) orally for 60 days.

Group IV: Rats model of chronic stress-induced depression received

combination of therapeutic dose of Duloxetine (30 mg/kg /day) and (400 mg/kg

/day) of omega 3 orally for 60 days.

Experimental protocols of chronic stress-induced depression:

The rats of group II, III and group IV were received a variety of stressors

according to Wang et al.( 2005) and Yang et al. (2006 ) for 30 days before

starting the treatment and the same method continues during the period of the

experiment. The following stressor were used to induced depression, tail node

for 1 min, cold water swimming at 4°C for 5 min, heat stress at 45°C for 5 min,

water deprivation for 24 h, food deprivation for another 24 h, 12-h inverted

light/dark cycle (8:00 a.m. lights off and 8:00 p.m. lights on), paw electric shock

(electric current 1.0 mA10 s, every 1 min, lasting 10 s, 30 times).



269

At the end of the experimental period (two months) and after overnight

fasting, blood samples were obtained from sinus orbitus vein of each rat after

ether inhalation (Yang et al., 2006). The blood samples were allowed to clot at

room temperature before centrifuging at approximately 3000 rpm for 15

minutes. The serum was stored at -20° C until assayed for the biochemical

parameters. Then, all studied animals were sacrificed; the two testes of each rat

were excised, one of them was prepared for estimation of the markers for

oxidative stress and the other was prepared for histopathological examination.

Moreover isolation of both vas deferentia was done to evaluate the effect of

norepinephrine induced contractions in all groups.

B) Chemicals:

*Duloxetine Hydrochloride: Symbalta, (Duloxetine Hydrochloride 30 mg

oral capsule) was produced by the Eli Li Co. for Pharmaceuticals and Chemical

industries, Cairo, A. R. E.

**Omega-3: (1000 mg capsules) was produced by South Egypt Drug

Industries Company (SEDIC), Cairo, A. R.E.

***Norepinephrine ampoules 1mg/ml (USP). Ascorbic acid white

crystalline powder (Merk).

C) Biochemical oxidative parameters:

The testes were rinsed in ice-cold 0.175 M KCl /25 mM Tris–HCl (pH 7.4)

to remove the blood, minced in the same solution, and homogenized by means

of a homogenizer with a Teflon pestle. The testis homogenates were centrifuged

at 10,000 rpm for 15 min. The supernatants were then used for lipid

peroxidation determination, and antioxidant enzyme assays as follows:



270

a) Tissue Glutathione (GSH) Analysis: The reduced GSH content of testis

tissues was estimated according to the method described by Sedlak and

Lindsay (1968).

b) Tissue superoxide dismutase (SOD) and catalase (CAT) activity

determination: The SOD activity was measured by the inhibition of nitroblue

tetrazolium (NBT) reduction due to O2 generated by the xanthine/xanthine

oxidase system (Sun et al., 1988). One unit of SOD activity was defined as the

amount of protein causing 50% inhibition of the NBT reduction rate. The CAT

activity of tissues was determined according to the method of Sinha (1991). The

enzymatic decomposition of H2O2 was followed directly by the decrease in

absorbance at 240 nm. The difference in absorbance per unit time was used as a

measure of CAT activity. The enzyme activity was given in U/mg of protein.

c) Determination of malondialdehyde levels: The levels of malondialdehyde

(MDA) in homogenized tissue, as an index of lipid peroxidation, were

determined by a thiobarbituric acid reaction using the method of Yagi (1998).

d) Determination of protein content: The tissue protein content was measured

according to Cannon (1974) using bovine serum albumin as a standard.

D) Isolated rat vas deferens: according to Jurkiewicz et al.( 1969)The effect

of different doses of NE (2-32µg/ml) was studied on the amplitude of

contractions of the isolated rat vas deferens in all studied groups by injecting

the drug into the perfusion fluid.

E) The histopathological preparation:

For light microscopic examination, the testes were fixed in Bouin’s solution

for 48 h. Later, they were dehydrated in graded levels of ethanol, cleared in

xylene, and embedded in paraffin wax for sectioning. The 4-μm thick sections

were cut, mounted on glass slides, and stained with hematoxylin and eosin stain.

Masson’s trichrome stain also used, it give the collagen fibers blue colour, the

nuclei appeared blue black and the cytoplasm appeared red. In addition Periodic



271

acid-Schiff (PAS) reagent stain was used, it demonstrates the glycogen and

other periodate reactive carbohydrates appeared magenta and nuclei appeared

blue (Bancroft and Steven, 1996).

F) Immunohistochemical Study:

Proliferating cell nuclear antigen (PCNA) is an intranuclear polypeptide

that is involved in DNA replication, excision and repair. Its synthesis and

expression is linked to cell proliferation (Shivji et al., 1992). Since

spermatogenesis is a complex cell cycle of rapidly proliferating cells ending

with liberation of sperms, PCNA was used in this study to quantitatively

analyze spermatogenesis. Immunohistochemical staining was carried out using

primary antiserum to PCNA (Clone PC 10, DAKO A/S Denmark). The primary

antibody was diluted in Trisbufferd saline with a dilution of 1:50, as determined

by the data sheet. The sections were incubated with the primary antibody

overnight at + 4°C. The binding of the primary antibody was observed using a

commercial avidinbiotin- peroxidase detection system recommended by the

manufacturer (DAKO, Carpenteria, USA). A mouse monoclonal antibody was

applied in place of the primary antibody to act as a negative control. Sections

from the small intestine were used as a positive control. Then the slides were

stained with diaminobenzene (DAB) as the chromogen and counterstained with

hematoxylin (Elias et al., 1989).

Slides were examined under the light microscope with a magnification

X100. Then sections were evaluated for PCNA immunostaining. Microscopic

fields were chosen at random. Five fields per slide and five slides per animal

were evaluated. Only the basal germ cells of these tubules were counted,

because they are the cells where active DNA synthesis took place (Heller and

Clermont 1964). For each specimen, the mean ± SEM was calculated. Then,

the total PCNA positive cells for all groups were estimated accordingly.

G) Statistical analyses:



272

One-way analysis of variance (ANOVA) test followed by Student, s test

were used. The data obtained in the present study were expressed as mean

SEM for quantitative variables and statistically analyzed by using SPSS

program (version 17 for windows) (SPSS Inc. Chicago, IL, USA). P value<0.05

was considered statistically significant.

RESULTS

Biochemical Results:

In the current study, it was found that testosterone (ng/ml), LH (µg/ml) and

FSH (µg/ml) serum levels in control group was 7.81±1.7 , 2.76±0.34 and

13.78±0.7 respectively, while in Duloxetine treated rats (group III) significantly

decreased to reach 3.84±0.65 , 1.02±0.26 and 7.15±0.5 respectively when

compared with the control (P<0.001). In Omega 3 treated rats (group II) it gave

significantly increase in testosterone, LH and FSH serum levels when compared

to (group III). In Duloxetine and Omega 3 treated rats (group IV) were given a

statistically significant increase in the levels of blood testosterone, LH and FSH

when compared to group III (P<0.001) (Table 1).

Table (1): Mean (± SEM) serum levels of testosterone, LH and FSH in the studied groups.

Parameters

Groups

Testosterone

(ng/ml) LH (µg/ml)

FSH (µg/ml)

Control group 7.81±1.7 2.76±0.34 13.78±0.7

Omega-3 treated group 8.90±1.5 3.84±0.15 15.21±0.1

Duloxetine treated group 3.84±0.65* 1.02±0.26

* 7.15±0.5

*

Duloxetine &Omega-3 treated group 7.66±0.91**

2.51±0.16**

13.35±0.2**

Values are expressed as mean ± SEM. *

Test of significance between control and

Duloxetine -treated rats. ** Test of significance between Duloxetine -treated and



273

Duloxetine and Omega-3 treated rats.

0

2

4

6

8

10

12

14

16

Testosterone LH FSH

Control group

Omega-3 treated group

Duloxetine treated group

Duloxetine &Omega-3 treated

group

Fig. (1): Mean serum levels of testosterone, LH and FSH in the studied groups.

Biochemical oxidative parameters:

The testes contents of GSH, SOD and CAT activities were significantly

decreased in Duloxetine treated rats (group III) compared to those in control

(group I). As regards the testes MDA content in group III showed a highly

significant increase in their testes content of MDA as compared to that of

control rats (Table 2 and Figs.2&3). Whereas, group II and group IV were given

a statistically significant increase in the levels of GSH, SOD and CAT and a

statistically significant decrease in MDA levels were determined when

compared to group III.



274

Table (2): Mean (±SEM) testes contents of MDA, GSH, SOD and CAT in the studied

groups.

Parameters

Groups

MDA

(nmol/mg)

protein

GSH(

u/mg)

protein

SOD

(u/mg)

protein

CAT

(u/mg)

protein

Control group 1.89±0.07 6.03±0.01 2.41±0.11 430±2.39

Omega-3 treated group 0.52±0.04 7.91±0.04

3.89±0.23 495±1.14

Duloxetine treated group 3.97±0.66* 3.15±0.04

* 1.01±0.57

* 260±1.65

*

Duloxetine &Omega-3 treated

group

1.02±0.03**

5.68±0.15**

2.29±0.23**

425±2.47**

Values are expressed as mean ± SEM. * Test of significance between control and Duloxetine -

treated rats at p < 0.001. **

Test of significance between Duloxetine -treated and

Duloxetine&Omega-3 treated rats at p < 0.001

0

1

2

3

4

5

6

7

8

MDA GSH SOD

Control group



Duloxetine & Omega-3

treated group

Fig. (2): Mean testes contents of MDA, GSH and SOD in the studied groups.



275

0

100

200

300

400

500

CAT

Control group

Omega-3

treated group

Duloxetine

treated group

Duloxetine &


Fig. (3): Mean testes contents of CAT in the studied groups.



276

Isolated rat vas deferens:

Duloxetine pretreatment (two months) inhibited the contractile response

of vas deferens to norepinephrine induced contractions, which was found to be

statistically significant as compared to that of control. Whereas, Omega-3 and

Duloxetine &Omega-3 treated groups were found to be statistically significant

increase in norepinephrine induced contractions (P < 0.001) when compared to

that of group III (table 3 & Fig3: a, b, c & d).

Table (3): Comparison between the Mean (±SEM) effects (cm) of NE (2-

32µg/ml) on vas deferens of rats isolated from the studied groups.

Doses

Groups

NE

2g

/ml

NE

4g

/ml

NE

8g

/ml

NE

16g

/ml

NE

32g

/ml

Control group

0.93±0.

047

1.94±0.

089

2.79±0.

052

2.96±0.

076

3.98±0.

049


1.89±0.

84

2.97±0.

091

3.75±0.

063

3.98±0.

045

4.99±0.

018


0.35±0.

034*

0.77±0.

042*

1.32±0.

047*

2.11±0.

047*

2.43±0.

049*

Duloxetine and Omega-3 treated group

0.85±0.

44*

1.73±0.

033**

2.48±0.

040**

2.81±0.

031**

3.85±0.

043**

Values are expressed as mean ± SEM. * Test of significance between control and

Duloxetine -treated rats. **

Test of significance between Duloxetine -treated &

Duloxetine and Omega-3 treated rats.



277

Fig.(4): Mean effect of NE (2-32µg/ml) on vas deferens of rats isolated

from control (a), group treated with Omega-3 ( b) , Duloxetine (c) and

Duloxetine and Omega-3 (d) for two months.

The results of the histopathological examination:

The histopathological examination of transverse sections of testes of

groups I (control) and II (omega 3 treated group) are similar. The testes are

covered by normal thin testicular capsule and it is consisted of several uniformly

arranged seminiferous tubules. The seminiferous tubules are surrounded by thin

basal lamina and lined by different stages of spermatogenic cells, spermatogonia

A, spermatogonia B, primary spermatocytes, spermatides and Sertoi cells in-

between. These cells form 6 to7 rows .The mature spermatozoa present in the

lumina. The seminiferous tubules are separated from each other by interstitial

tissues containing blood vessels and interstitial cells of Leydig (Figs.5, 6:

A&B). A normal collagen fibers distribution can be seen in the testicular

capsule, basal lamina and interstitial tissues (Fig.7: A&B). By using PAS



278

reaction, it is showing that thin basement membrane surrounding the

seminiferous tubule (Fig.8: A&B).

The histopathological finding in testes of group III (Duloxetine treated group)

showed marked signs of cellular degeneration and necrosis. There were great

depletion of germ cells and Sertoli cells. The germinal epithelium of affected

tubules form only 2 to 3 rows of disorganized, disrupted germ cells and it was

difficult to differentiate between them, whereas most of these cells had pyknotic

nuclei. There were no sperms observed in the lumina of some seminiferous

tubules (Figs.5, 6: C). In addition, the seminiferous tubules appeared widely

separated from each other. On the other hand, the interstitial cells of Leydig

showed the same degeneration and necrosis, whereas most of them appeared

pyknotic (Fig.6: C). Marked increase of the collagen fibers deposition in the

testicular capsule, around the blood vessel of tunica vasculosa , the basal

lamina and in the interstitial tissues were also present (Fig.7: C).By PAS

reaction the basement membrane of the seminiferous tubule appeared thick with

deposition of PAS positive materials in between the seminiferous tubule (Fig.8:

C).

Moderate regeneration and improvement were observed in the

histopathological examination of testes of group IV (Duloxetine and omega3

treated group) whereas, some seminiferous tubules contain many rows of

different stages of spermatogenic cells and Sertoi cells in-between.

In spite of increment of the rows of the spermatogenic cells, the cells appeared

with deeply stained nucleoli and difficult to differentiate between them. Small

amount of mature spermatozoa appeared in the lumina. In addition, some

seminiferous tubules still had degenerated spermatogenic cells containing

pyknotic nuclei (Figs.5&6: D). Less collagen fibers deposition could be seen in

the testicular capsule, in the basal lamina and in the interstitial tissues when



279

compared with group III (Fig.7: C and D). PAS positive materials deposition

was still present in between the seminiferous tubules (Fig.8: D).

Immunohistological Results:

The positive PCNA immunostaining spermatogenic cells (deeply brown in

color) included mainly the spermatogonia and primary spermatocytes. The

positive immunostaining appeared quite evident in the nuclei of the

spermatogenic cells of group I (control) and in group II (Omega3 treated group)

which are apparently more packed and exceeding the basal germ cells by 2 to 3

row. It was less evident in the testes of group IV (Duloxetine and Omega3

treated group) and least in the testes of group III (Duloxetine treated group)

(Fig.9). Application of ANOVA test revealed that there were highly significant

decrease (P < 0.001) in number of positive PCNA immunostaining cells in

group III (Duloxetine treated group) when compared to the control . Addition of

Omega 3 in group IV increase the number of positive PCNA immunostaining

cells and make a significant increase (P < 0.001) when compared to the group

III (Duloxetine treated group)( Fig.10 and table 4).



280

Fig.5: Photomicrograph of transverse sections of the studied testes demonstrating:

A; Control and B; Omega 3 treated group both contained normal

seminiferous tubules surrounded by basal lamina ( arrow)and having different

stages of spermatogenic cells and Sertoli cells in-between. The mature

spermatozoa (S)appeared in the lumina. Notice also the presence of normal

interstitial cells of Leydig (g) in between the seminiferous tubules.

C; Duloxetine treated group notice; many degenerated seminiferous tubules

(1, 2, 3, 4) with presence of wide spaces (*) between them.

D; Duloxetine and omega3 treated group show presence of improved

seminiferous tubules containing many rows of different stages of spermatogenic

cells while some degenerated seminiferous tubules (1,2) still present.

(Hx. & E.; X100).



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Fig.6:Photomicrograph of transverse sections of the studied testes demonstrating:

A; Control and B; omega3 treated group both have seminiferous tubule

contained from 6 to7 rows of different stages of normal spermatogenic cells.

spermatogonia A (a), spermatogonia B (b), primary spermatocytes (m) ,

spermatides (d) and the Sertoli cells(r) in-between. The mature spermatozoa (S)

appeared in the lumina.

C; Duloxetine treated group notice; the seminiferous tubule contained only 2

to 3 rows of degenerated spermatogenic cells (P) which difficult to differentiate

between them. Notice the presence of pyknotic interstitial cells of Leydig (g).

D; Duloxetine and omega3 treated group show the seminiferous tubule

contains from 6 to7 rows but it difficult to differentiate between the different



282

stages of it ,because it have deeply stained nucleoli. Some mature spermatozoa

(S) appeared in the lumina (Hx. & E.; X400).

Fig.7: Photomicrograph of transverse sections of the studied testes

demonstrating:

A; Control and B; omega3 treated group show normal distribution of

collagen fibers in the testicular capsule(E) vessels of tunica vasculosa (V) ,

basal lamina(arrow) and interstitial tissues (i) .

C; Duloxetine treated group show marked increase of the collagen fibers

deposition in the wavy testicular capsule(E), around a blood vessel in the

tunica vasculosa (V) ,the basal lamina (arrow) and in the interstitial tissues (i).

D; Duloxetine and omega3 treated group show less collagen fibers

deposition in the testicular capsule (E), in the basal lamina (arrow) and in the

interstitial tissues (i) (Masson’s trichrome x 100).



283


demonstrating:

A; Control and B; omega3 treated group showing a thin basement membrane

surrounding a seminiferous tubule (arrow).

C; Duloxetine treated group showing thickened basement membranes of the

seminiferous tubules with PAS positive materials deposition in between

seminiferous tubule (arrows).

D; Duloxetine and omega3 treated group show thin basement membranes and

less PAS positive materials deposition in between seminiferous tubule (arrows).

|(PAS x100).



284


demonstrating:

A; Control showing positive immunostaining (brown nuclear reaction) in all

the nuclei of the basal germ cells of the seminefirous tubules.

B; Omega3 treated group showing the positive immunostaining cells which

are apparently more packed and exceeding the basal germ cells by 2 to 3 row .

C; Duloxetine treated group showing few positive immunostaining germ cells

(arrow).Notice the presence of wide spaces (*) between the seminiferous

tubules.

D; Duloxetine and omega3 treated group show that many positive

immunostained basal germ cells appear in some seminiferous tubules (1) and

other tubules contained 2 to 3 rows of positive immunostaining germ cells (2).



285

Notice that the wide spaces (*) are still present between the seminiferous

tubules. (PCNA immunohistochemical staining X 100).

Table (4): Mean (±SEM) of the positive immunostaining nuclei of the

seminiferous germ cells in all studied groups.

Parameter

Groups

positive immunostaining seminiferous

cells

Control group 64.24±0.07

Omega-3 treated group 79.52±0.04

Duloxetine treated group 20.97±0.66*

Duloxetine & Omega-3 treated group 45.02±0.03**

Values are expressed as mean ± SEM. * Test of significance between control

and Duloxetine -treated rats at p < 0.001. **

Test of significance between

Duloxetine -treated and Duloxetine&Omega-3 treated rats at p < 0.001.

0

10

20

30

40

50

60

70

80

Control group Omega-3

treated group

Duloxetine

treated group

Duloxetine &

Omega-3

treated group

Germ cells

Control group


Duloxetine treated

groupDuloxetine & Omega-3

treated group

Fig. (10): Mean of the positive immunostaining nuclei of the seminiferous

germ cells in all studied groups.

DISCUSSION

The present study evaluated the effects of Duloxetine on the testes and its

function. The histopathological changes were in concomitant with hormonal and



286

biochemical changes. There were significant decrease in the testosterone, LH

and FSH levels with significant increase of testes content of MDA, while testes

content of GSH, SOD and CAT activities significantly decreased. Furthermore,

Duloxetine pretreatment produced a dose dependent decrease in the amplitude of

norepinephrine induced contractions in isolated rat vas deferens. Also, by light

microscopic examination the testes of group III (Duloxetine treated group)

showed massive degenerative changes in the form of great depletion of germ

cells and Sertoli cells. The germinal epithelium of affected tubules form only 2

to 3 rows of disorganized, disrupted germ cells and it was difficult to

differentiate between them, whereas most of these cells had pyknotic nuclei.

There were no sperms observed in some lumina. The attributed causes for the

previous pathological and biochemical changes were that Duloxetine lead to

disturbance of the hormonal axis and it may expose the testes to oxidative stress,

production of free radicals and peroxidative damage. This suggestion goes with

Aitken et al. (1989) who reported that oxidative stress results in defective

spermatogenesis, with the subsequent decrease in sperm production and the

release of immature ROS producing spermatozoa into the seminiferous tubules.

Aitken and Krausz (2001) added that the spermatozoon has a high content of

polyunsaturated fatty acids within the plasma membrane and a low

concentration of scavenging enzymes within the cytoplasm. Therefore, it is

susceptible to the peroxidation in the presence of elevated ROS seminal levels

and oxidative stress induces DNA damage in both the mitochondrial and nuclear

genomes.

Also, in accordance to the current results, Afify et al. (2010) reported that

GSH levels significantly decreased, while MDA levels significantly increased in

rats testes treated with antidepressant amitriptyline which indicate that

antidepressants may cause oxidative stress in the testes. Furthermore, Soghra et



287

al. (2008) showed that the antidepressant amitriptyline produced decrease of the

testosterone level leads to sexual dysfunction and infertility. Also, in agreement

with our results Weydt et al (2011) reported that Duloxetine can produce a

relevant dysfunction in the reproductive endocrine axis which can produce

hypogonadism and gynecomastia. In addition, França et al.( 2005) and

Turner,( 2007) have shown the importance of testosterone to the qualitative and

quantitative maintenance of spermatogenesis, so reduction of plasma or intra-

testicular levels of testosterone may have led to testicular degeneration. In

addition, Ozyavuz et al. (2004) mentioned that, rat isolated vas deferens shown

that sertraline, antidepressant, pretreatment inhibits contractile responses to

norepinephrine, KCl, serotonin and electrical field stimulation

In the present study, Omega-3 fatty acid is found very effective in protection

from Duloxetine induced toxicity in testes and in the isolated vas deferens. It

leads to marked elevation of the testes contents of GSH, SOD and CAT.

Moreover improvement of testosterone, LH and FSH with decrease of MDA are

noticed when compared to Duloxetine treated group. Also, moderate

regeneration and improvement in the histopathological examination of the

seminiferous tubules of the testes of group IV (Duloxetine and omega 3 treated

group) are present. Some seminiferous tubules contain many rows of different

stages of spermatogenic cells and Sertoi cells in-between. These improvements

are attributed to the powerful antioxidant effects of the omega 3. This

suggestion was supported by Spencer et al.( 2009) who cited that Omega-3

fatty acids attenuated cancer cells growth and induce apoptosis in a variety of

human cancer cell lines derived from colonic, pancreatic, prostate, and breast

cancer. In addition, findings of Klaunig and Kamendulis,( 2004) and

saravana et al .(2011) reported that the chemotherapeutic agents induced

testicular toxicity were effectively protected by omega 3 fatty acid. Moreover,

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288

Ismail et al.( 2011) reported that Omega-3 stimulates spermatogenesis and

increase the activity of Sertoli cells. In a normal situation, the cellular

antioxidant mechanisms present in almost all tissues and their secretions are

likely to quench those reactive oxygen species (ROS) and protect against

oxidative damage (Vertuani et al., 2004 and Dalle- Donne et al., 2006).

Hence the testicular tissues are very sensitive to ROS effects. In the present

study, administration of Omega-3 reversed these alterations in the enzyme

activities and brought the levels of all three enzymes near to the control levels.

These results indicate that Omega-3 has a primary role in mediating the

scavenger action. It also has a beneficial effect for spermatogenesis following

Duloxetine induced testicular damage by decreasing germ cell apoptosis and

oxidative stress. The results of the present study were in agreement with Liangli

(2001), who reported that the antioxidant properties of Omega-3 fatty acids

normalized and protected against the decrease in sperm count, motility and

viability as well as, the sperm abnormalities. In addition Omega-3 fatty acids

also restored the antioxidant activities (reduced the malondialdehyde level,

increased the reduced glutathione, superoxide dismutase and glutathione

peroxidase levels) that were harmfully affected by antidepressants

administration. Freeman et al. (2001) and Saravana et al. (2011) added that

after Omega-3 administration in mice, the decreased levels of antioxidant

constituents were restored in the testes tissues. Its antioxidant effects provide a

defense mechanism through three levels of protection- prevention, interception

and repair. Hales et al. (2005) & Bansal and Bilaspuri (2009) added it has

been found that Omega-3 had protective role on spermatogenesis against

antidepressants induced cell damage which is in agreement with the present

study.



289

In the resent study, in group III, prominent deposition of collagen fibers in

the testicular capsule, the basal lamina and in the interstitial tissues, in addition

to presence of PAS positive materials in between the seminiferous tubules were

attributed to the oxidative stress. This explanation was supported by the finding

of Bacon and Britton (1989) who reported that formation of hydroxyl radical

and other highly reactive oxidizing molecules in biological system led to lipid

peroxidation. The latter caused oxidative damage to proteins and nucleic acids.

The end results of these reactions led to increase of collagen and ground

substance formation. Also, the presence of wide spaces between the

seminiferous tubules suggesting the presence of interstitial oedema, the results

of both Lucesoli et al. (1999) and Patil and Balaraman (2009) supported this

finding.

Administration of Omega3 with Duloxetine led to less deposition of

collagen fibers in the same areas and less PAS positive materials deposition was

present due to antioxidant effect of Omega-3. Gonzalez et al. (1993) mentioned

that vitamin E resides in the lipid domain of biological membranes and plasma

lipoprotein, where it prevented lipid peroxidation of polyunsaturated fatty acids

(PUFA). Also, Menna ,(2009) found that the basement membranes of the

seminiferous tubules of testes exposed to oxidative stress appeared thickened

with deposition of PAS positive material in the interstitial spaces .The author

observed decrease of these deposited material after administration of antioxidant

resveratrol .

By the results of the present study, it is well known that Duloxetine have

deleterious effects on the spermatogenesis and administration of Omega3 with it

improve the process of dividing spermatogenic cells. Histopathological

assessments were methods for measurements of testicular health, but generally

they are neither quantitative nor sensitive enough to detect the early toxicity or



290

recovery. Recently, an immunolabeling cell with proliferating cell nuclear

antigen (PCNA) has been used to identify proliferating spermatogonia;

however, there have been no systematic attempts to quantify these changes.

(PCNA) have developed a sensitive, reliable and quantitative assay using

immunohistochemistry on formalin fixed, paraffin embedded rat testes to assess

the degree of proliferation-related toxicity (D'Andrea et al., 2008). Bravo and

Mac Donald ( 1985) previously reported that PCNA is a stable protein and can

be detected in quiescent cells 24 to 48 h after cells stop dividing, so it used in

the current study to evaluate, by counting , the normal dividing spermatogenic

cells. Also, Hall et al., (1990) had previously observed that PCNA staining cells

were in the majority of the spermatogonia and no staining was present in

spermatids or sperms in the human testis.

In the present study, PCNA immunostaining cells of the testes of

Duloxetine treated group demonstrated few positive immunostaining basal

spermatogenic cells with highly significant decrease when compared to the

control group. These finding confirmed that the oxidative stress of the germinal

epithelium led to depletion of the active DNA contents in these dividing cells in

this group which leading, as an end result, to depletion of spermatozoa. This

suggestion was in agreement with (Agarwal and Said, 2003) who reported that

oxidative stress in the testis has shown to cause peroxidative damage to integrity

of sperm DNA which has become an area of focus in male infertility studies.

Similar study by Sakkas et al.( 2002) have identified damage to DNA,

characterized by a high incidence of base modification, DNA fragmentation,

chromatin cross-linking and DNA strand breaks in spermatozoa of infertile men.

The DNA damage that was observed in those studies was attributed to high

levels of ROS in spermatozoa. Also, Duran et al. (2002) and Meseguer et al.

(2008) have shown that DNA damage of sperm cell can cause low quality of



291

sperm and fertilization problems since spermatozoa requires intact DNA during

fertilization process. Oxidative damage to mitochondrial DNA (mtDNA) is also

known to occur in spermatozoa and other aerobic cells that are rich in

mitochondria. Multiple mtDNA deletions in spermatozoa could arise through a

free radical driven event occurring at the spermatogonial cell stage and has been

linked to reproductive failure in some men. Furthermore , Schlat and

Weinbauer (1994) confirm the results of the present study, the authors cited

that hormonal withdrawal induced a decrease in the number of PCNA-positive

cells in adult rats, hamsters and monkeys.

In conclusion, Duloxetine administration induces quantitative and

qualitative alterations in the hormonal levels, oxidative parameters and process

of spermatogenesis, leading to testicular degeneration. Antioxidant, Omega-3,

preserve adequate functions of the testes cells against disturbances caused by

Duloxetine induced oxidative stress.

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http://www.ncbi.nlm.nih.gov/pubmed?term=Weinbauer%20GF%5BAuthor%5D&cauthor=true&cauthor_uid=7995658



292

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GSH CAT دأدي أؼب إن إسرفبع يسزب LH, FSHف يسزبد شي انزسزسزش،

SOD (P ˂ 0.001 ) إخفبع ف يسزي إن MDA أدي إن إخفبػب كب عذ أ انذكسز

أعط إػبفخ . نهفئشا ف انزقهظبد انبرغخ ع انسادسبن ف انعبء انبقم( P ˂ 0.001) كجشا

أدي إن عاليبد قهخ انذهكسز كب عذ أ. زح انزقهظبد ان صبدح راد دالنخ إحظبئخ ف 3ايغب

. انخالب خش يع إسزضاف كجش ف انخالب انغشصيخ خالب سشرن ف األبثت انخ انزؼشسح

نغشصيخ ،رعطهذ كب ي انظعت انزفشق ثب ، ف ح أ انحابد قذ إخزهذ طفف انخالب ا

ثبإلػبفخ إن إخفبع كجشر دالنخ إحظبئخ ف عذد انخالب انبعخ . انخ نى رك يعدح

(PCNA) ( P ˂ 0.001 )قذ أعط إػبفخ ايغب . انز رفش أدنخ عهى رعشع انخظز نألكسذح

دذ انعزذل انزحس ف فحض األسغخ حش أ ثعغ األبثت انخ إحزد عهى انعذذ ان انزظ 3

ي طفف انخالب انخ خالب سشرن ف يشاحم يخزهفخ ي ان كب ظشد انحابد انخ

PCNA (P ˂ 0.001 )انبػغخ كب نحع صبدح راد دالنخ إحظبئخ ف عذد انخالب انبعخ

.ثبنقبسخ ثبنغعخ انضبنضخ

دهكسز عشع انخظز نؼغؾ االكسذح يب ؤدي إنى إخزالل ف يسزبد انشيبد : اإلستىتبج

عم عه انحفبظ عهى ظبئف 3إػبفخ أيغب . ،دالالد األكسذح عهخ رك انحابد انخ

.ل انذهكسزكبفخ نهخظخ ػذ اإلػطشاثبد انبعخ ع رب



300

التغيرات الحيىكيميبئية و الهستىلىجية والهستىكيميبئية المىبعية لخصي الفئران الببلغة

في ومىرج 3المعبلجة ببلذيلىكستيه هيذروكلىرايذ والذور الىقبئي المحتمل لالوميجب

مستحذثة اإلكتئبة الفئران

سحر بذر الذيه *

ور عبذ العبطيألفث أنو **

( ثبد) كهخ انطت عبيعخ األصش -انزششح **انفبسيبكنغ *قس


نؼبدح نالكزئبة ي ث رهك األدخ انز رزسجت ف آصبس سبيخ عهى انكضش ي رعزجش انعقبقش ا

انذهكسز احذ ي يضجطبد إيزظبص انسشر . األعضح خبطخ انغبص انزبسه انزكشي

ي يؼبداد 3عزجش ايغب . ف سزخذو نعالط انعذذ ي االػطشاثبد االكزئبثخ. انسادسبن

. عجبسح ع أحبع دخ ػشسخ غش يشجعخ. كسذ انقخألا

نهزذنم عهى انزغشاد انجكبئخ انسغخ انبعخ ف خظ انفئشا : الهذف مه البحث

.3انجبنغخ ثعذ إسزحذاس اإلكزئبة صى ربل انذهكسز أؼب رقى انزأصش انقبئ انحزم نأليغب

رى رقسى صب ي ركس انغشرا انجؼبء انجبنغخ ف أسثع يغعبد : البحثالمىاد وطرق

( ػبثطخ) انغعخ األنى . يزسبخ قذ رى إسزحذاس اإلكزئبة ف انغعخ انضبخ انضبنضخ انشاثعخ

، (و / كغ / يهغ 400) 3رهقذ انبء انقطش ع ؽشق انفى ، انغعخ انضبخ رزبل أيغب

انغعخ انشاثعخ رهقذ ( و / كغ / يهغ 30) انغعخ انضبنضخ رزبل انذهكسز ذسكهسذ

. نذح شش( و / كغ / يهغ 400) 3يع أيغب ( يب/ كغ / يهغ 30) انذهكسز ذسكهسذ

قذ رى قبط عاليبد , LH, FSHف بخ انفزشح انزغشجخ رى رقى شيبد انزكسح انزسزسزش

عالح عهى رنك،رى فحض األسغخ ثبنكشسكة انؼئ ثعذ طجبغزب . األكسذح ف انخظخ

ثبنبركسه األس رفبعم شف حغ انجشدك طجغخ يبص انضالص كزنك طجبغزب

كب أعشي رقى رقهظبد (PCNA) . حثظجغخ سزكبئخ يبعخ نؼبداد أخ انخالب انزكبصش

.انعبء انبقم نهفئشا ف كم انغعبد

,LHأظشد انزبئظ أ انذهكسز رسجت ف إخفبع كجش ف شي انزسزسزش، : الىتبئج

FSH . عالح عهى إخفبعGSH CATSOD إخفبػب كجشا (P ˂ 0.001 ) يع صبدح

ان صبدح راد دالنخ إحظبئخ 3قذ أعط إػبفخ ايغب . MDA (P ˂ 0.001) رشكضف

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