https://doi.org/10.52973/rcfcv-e33288
Received: 03/07/2023 Accepted: 01/08/2023 Published: 20/08/2023
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Revista Científica, FCV-LUZ / Vol. XXXIII, rcfcv-e33288
ABSTRACT
This study investigated the hormonal, inflammatory, oxidant–
antioxidant, and histopathological effects of exogenous Melatonin
administration on Spermatogenesis in rats' chronic unpredictable
stress model (CUSM). In the study, stress caused a decrease in follicle
stimulating–hormone (FSH), luteinizing hormone (LH), Testosterone,
Melatonin, Glutathione (GSH), Glutathione peroxidase (GSH–Px),
catalase, interleukin 10 (IL–10) levels and motility, and an increase in
Corticosterone, nuclear factor kappa beta (NF–kB), tumor necrosis
factor–alpha (TNF–α), interleukin 1 beta (IL–1β), interleukin 6 (IL–6),
abnormal sperm, dead/live sperm ratio and exogenous Melatonin
reduced inammatory cytokines and oxidative stress and improved
spermatological parameters (P<0.05). Melatonin also partially
corrected stress–induced changes in testicular morphology. As
a result, using Melatonin in rats with CUSM may be effective in
improving spermatological parameters through anti–inammatory
and antioxidant mechanisms.
Key words: Chronic stress; spermatogenesis; Melatonin;
antioxidant; anti–inammatory cytokines
RESUMEN
Este estudio investigó los efectos hormonales, inamatorios, oxidantes–
antioxidantes e histopatológicos de la administración exógena de
melatonina sobre la espermatogénesis en el modelo de estrés crónico
impredecible (CUSM) de ratas. En el presente estudio, el estrés provocó
una disminución de los niveles de hormona foliculoestimulante (FSH),
hormona luteinizante (LH), testosterona, melatonina, glutatión (GSH),
glutatión peroxidasa (GSH–Px), catalasa, interleucina 10 (IL–10) y
motilidad, y un aumento de la corticosterona, factor nuclear kappa
beta (NF–kB), factor de necrosis tumoral alfa (TNF–α), interleucina 1
beta (IL–1β), interleucina 6 (IL–6), espermatozoides anormales, relación
espermatozoides muertos/espermatozoides vivos y la melatonina
exógena redujeron las citocinas inamatorias y el estrés oxidativo y
mejoraron los parámetros espermatológicos (P<0,05). La melatonina
también corrigió parcialmente los cambios inducidos por el estrés en la
morfología testicular. Como resultado, el uso de melatonina en ratas con
CUSM puede ser ecaz para mejorar los parámetros espermatológicos
a través de mecanismos antiinamatorios y antioxidantes.
Palabras clave: Estrés crónico; espermatogénesis; melatonina;
antioxidante; citoquinas antiinamatorias
Effect of exogenous Melatonin administration on Spermatogenesis in
chronic unpredictable stress rat model
Efecto de la administración exógena de Melatonina sobre la Espermatogénesis en un modelo de rata
con estrés crónico impredecible
İshak Gökçek
1
* , Leyla Aydın
2
, Mustafa Cellat
1
, İlker Yavaş
3
, Tuncer Kutlu
4
1
Hatay Mustafa Kemal University, Faculty of Veterinary Science, Department of Physiology. Hatay, Turkey.
2
Ankara Yıldırım Beyazıt University, Faculty of Medicine, Department of Physiology. Ankara, Turkey.
3
Hatay Mustafa Kemal University, Faculty of Veterinary Medicine, Department of Reproduction and Articial Insemination. Hatay, Turkey.
4
Hatay Mustafa Kemal University, Faculty of Veterinary Medicine, Department of Pathology. Hatay, Turkey.
*Corresponding author: ishakgokcek@hotmail.com
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INTRODUCTION
Stress decreases semen quality male reproductive system [1, 2].
It causes many effects, such as a decrease in testicular antioxidant
capacity [3], increases in testicular apoptosis [1], and damage to the
blood testicular barrier [4] among others.
Reproductive system pathologies are an actual problem for all species
[2, 5, 6, 7]. Stress is known to cause infertility [1, 2, 4]. Infertility is a
global health problem affecting approximately 15% of couples [8], and
male infertility accounts for 40–50% of all infertility cases [9]. In studies
to explain the etiopathogenesis of infertility in chronic stress, it has
been argued that chronic stress may cause inhibition of Testosterone
production from Leydig cells by glucocorticoid [10], decrease in the
expression of some genes involved in Testosterone synthesis [11],
inammation and systemic or local oxidative stress [12].
Melatonin, the main indolamine the pineal gland produces, is small and
highly lipophilic, easily crosses biological membranes, and reaches all
cell parts [13]. It has been reported that Melatonin, which easily crosses
the blood–testicular barrier and has very low toxicity, is protective in
male reproductive health [1]. Exogenous Melatonin has antioxidant,
anti–inammatory, and antiapoptotic effects in many tissues, including
the testes [13, 14]. Decreased Melatonin levels in stressful situations
contribute to stress–induced infertility with different pathways [15].
This study aimed to investigate the possible effects of exogenous
Melatonin use on Spermatogenesis and the possible mechanisms
of these effects in terms of hormonal, inammatory, and oxidant–
antioxidant in rats with chronic unpredictable stress model (CUSM)
MATERIAL AND METHODS
Ethics, study design, and animals
The study was approved by the Hatay Mustafa Kemal University
Animal Experiments Local Ethics Committee (HADYEK) ethical committee
decision numbered 2021/03/06. Animal care and experimental protocol
complied with the National Institutes of Health (NIH) Guide for the Care
and Use of Laboratory Animals. (12 h light; 12 h darkness; 24 ± 3°C).
During the experiment, the rats (Rattus norvegicus) were fed with
commercial pellet food and tap water ad libitum.
In the study, 40 male rats (Wistar albino) (40–50 days old, 180–200g)
were used. Animals were divided into ve groups Control (C) (n=8),
Vehicle (V) (n=8), Stress (S) (n=8), Melatonin (M) (n=8), and Stress +
Melatonin (SM) (n=8).
Stressors (social stress, cage tilt, without sawdust, damp sawdust,
foreign object, cage changing, restricted food access, sound of predator
noise) were applied to S and SM groups for eight weeks daily [16, 17].
Melatonin (Alpha Aesar>99) (10 mg·kg
-1
) administration to M and SM
groups was performed at 17:00 for 21 d between the sixth and eighth
weeks as daily orally [18, 19]. Group V was given 10% Dimethylsulfoxide
(DMSO) as Melatonin solvent [20]. At the end of the eighth week, rats
were anesthetized with Xylazine Ketamine, and the animals were
sacriced by intracardiac blood collection. Also, spermatological
analyzes were performed by collecting epididymal tissue samples.
Serum and testicular tissue analysis
Blood was collected from animals under Xylazine Ketamine anesthesia
and centrifuged (Nüve, Nf 800R, Turkey) at 3000 G for 10 min [21]. In
the obtained blood serum samples, Follicle stimulating hormone (FSH),
Luteinizing hormone (LH), and Testosterone levels were measured by
Radioimmunoassay (RIA); Corticosterone, Melatonin, Tumor necrosis
factor alpha (TNF–α), Interleukin 1 beta (IL–1β), Interleukin 6 (IL–6) levels
were measured with commercial Enzyme–linked Immunosorbent Assay
(ELISA) kits (Bioassay Technology Laboratory) and microplate reader
(Erba Manheim, Lisascan EM, Czech Republic).
For oxidative stress analysis, testis tissues were weighed (Weightlab
WL–303, Turkey) and transferred to glass tubes and then homogenized
by adding 1/10 (w/v) Tris Buffer (pH:7.4) to them. After the homogenates
were centrifuged (Nüve, Nf 800R, Turkey) at 3000 G for 60 min, the
supernate part was separated. Malondialdehyde (MDA), Glutathione
(GSH) levels, Glutathione peroxidase (GSH–Px), and catalase activities
were measured from the supernatant in tissue with a spectrometer
(Shimadzu, UV–1700, Japan) [21].
For testicular cytokines analysis, testes were homogenized by diluting
with Phosphate–buffered saline (PBS) (pH 7.4) 1/10 (w/v) and centrifuged
(Nüve, Nf 800R, Turkey) at 16000 G +4°C for 15 min, and TNF–α, IL–1β,
IL–6, Interleukin 10 (IL–10), Nuclear factor kappa beta (NF–kB) levels
were measured on tissue serum samples with commercial ELISA kits
(Bioassay Technology Laboratory, Zhejiang, China) and microplate
reader (Erba Manheim, Lisascan EM, Czech Republic).
Spermatological analysis
After the testicles were taken out and cleared of fatty tissues, the
cauda epididymis was separated. Left and right cauda epididymis, each
separately, in a petri dish (Isolab 60 × 15mm) with a 1 mL (PBS containing
0.1 M Glucose and 0.1 M Na–Citrate) diluent pre–warmed to 37°C and
under a stereo microscope (Euromex, Nexius Zoom, Netherlands) with
a scalpel and it was cut with scissors and incubated for 10 min in an
incubator (Nüve, N300, Turkey) at 37 °C in order for the spermatozoa to
reach the extender. After incubation, sperm samples were examined to
determine spermatological parameters (motility, abnormal, dead–live
sperm). To determine motility, 20 μL of sperm content was taken from
each sample and placed on a slide, and 300 sperm cells were counted
on each slide at 37 °C at 400 magnication. In the count, the ratio of
motile spermatozoa making robust, smooth, and linear movement in
the forward direction and non–motile spermatozoa was calculated in
the area where motile spermatozoa were examined [22].
The Eosin–Nigrosin staining method was used for dead spermatozoa
and abnormal sperm analysis [23]. Fifty µL of the semen sample and
50 µL of Eosin–Nigrosin solution were mixed. A smear was prepared
from this mixture and allowed to dry on a hot oor at 37 °C. Dried
smears were examined under a microscope (Olympus CX31, Japan), at
400 times magnication. Those whose head part of the spermatozoon
did not receive dye were considered alive, and those whose heads
were dyed red were considered dead. Three hundred spermatozoa
were counted for each sample, and the result was determined as %
dead spermatozoa. In abnormal sperm analysis, 300 spermatozoa
were counted in the counting area for each sample, and those with
abnormal morphology were expressed as a percentage.
Histopathological evaluation
Testicular samples were xed in 10% buffered formalin (pH 7.2–7.4).
After xation, it was reduced to 4 mm thickness and washed. It was
blocked in paran after being passed through graded alcohol (50, 80,
96 and 100%), xylol, and paran series according to routine methods.
Sections taken from the prepared four micron–thick tissue blocks cut
with a microtome blade (Pfm medical, Feather A35, United Kingdom)
FIGURE 1. Spermatological parameters. Values are Mean ± SEM and different letters
(a, b, c) indicated represent statistical signicance between groups (P<0.05)
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were rst thawn in a 37˚C water bath, then transferred to slides and dried
in an oven at 54˚C for 30 min. These sections were deparanized in
xylol, passed through 100, 96, 80, and 70 alcohol series, and stained with
Hematoxylin Eosin (H–E). They were passed through graded alcohol (80,
96 and 100%) and xylol again and closed with a coverslip with the help of
an adhesive (Entellan). They were examined under a light microscope
and their microphotographs (Olympus DP12, Japan) were taken.
Statistical analysis
Data were evaluated statistically. For all variables obtained,
parametric test assumptions were applied before the signicance
tests. Variables were analyzed with the Shapiro–Wilk test for normality
and the Levene test for homogeneity. One–way analysis of variance
(ANOVA) was then used to examine the difference between the variables
statistically. Tukey test was used as the Posthoc test for the variables in
which the difference between the groups was signicant. All statistical
analyses were conducted with a minimum margin of error of 5%. IBM
SPSS 23.0 software package was used in all statistical analyses.
RESULT AND DISCUSSION
Spermatological parameters
There was no statistical difference between the spermatological
parameters (motility, abnormal sperm, dead–live ratio) of the C, V,
and M groups. The S group showed decreased motility and increased
abnormal and dead–live ratios (compared to C, V, and M groups). In the
SM group, motility, abnormal sperm, and dead–live ratio were measured
as 63.12 ± 0.58, 12.38 ± 0.5, and 22.88 ± 0.44 (P<0.05 compared to the S
group). Spermatological parameters are shown in Fig. 1.
Serum hormone and cytokine levels
Serum FSH, LH, and Testosterone levels were similar in groups C
and V. It was similar in the M and S groups and signicantly lower than
in control. The lowest hormone levels were in the SM group (P<0.05).
Serum corticosterone levels of groups V and M were similar to
the control group. While there was a signicant increase in the S
group compared to the control, Melatonin treatment in the SM group
signicantly decreased it compared to the stress group (P<0.05). The
highest serum Melatonin level was measured in group M (P<0.05).
While the serum Melatonin levels of the V and SM groups were similar
to those of the C group, a statistically signicant decrease was
observed in the serum Melatonin levels in the S group.
Serum TNF–α and IL–6 levels were highest in the S group compared
to the other groups (P<0.05), and there was no statistically signicant
difference in the C, V, M, and SM groups. Moreover, serum IL–10 levels
were signicantly lower in the S group than in the other groups (P<0.05).
No statistically signicant difference existed in the C, V, M, and SM
groups. Serum hormone and cytokine levels are shown in Fig. 2.
Testicular tissue oxidative stress, and cytokine levels
Testicular tissue MDA levels were similar in the C, V, M, and SM
groups but were higher in the S group than in the other groups
(P<0.05). GSH levels were also similar in the C, V, M, and SM groups
but signicantly decreased in the S group compared to the other
groups (P<0.05). GSH–Px and catalase levels were not statistically
different in the C, V, and M groups but signicantly lower in the Sgroup
and higher in the SM group than the S group (P<0.05).
FIGURE 2. Serum hormone and cytokine levels. Values are Mean ± SEM and different letters (a, b, c) indicated represent statistical signicance between groups (P<0.05)
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Testicular pro–inammatory cytokine (NF–κB, TNF–α, IL–1β, and
IL–6) and anti–inammatory cytokine (IL–10) levels of the V, M, and
SM groups were similar to that of the control group. In group S, there
was an increase in proinammatory cytokine levels and a decrease in
anti–inammatory cytokine levels compared to other groups (P<0,05).
Testicular tissue oxidative stress and cytokine levels are shown in Fig. 3.
Histopathological ndings
In the current study, when the histological sections of the
seminiferous tubules (ST) of the C, V, and M groups were examined
under 20 and 100 µm magnication, it was seen that the ST were
regular in terms of shape, structure and mature sperm density in
the tubule lumen. There was no significant difference between
them. Morphologically, in the S group, deformations in seminiferous
tubule shape and structures and decreases in sperm density in the
seminiferous tubule lumen were observed compared to the control
group. In the SM group, the shape and structure of the ST were similar
to the control group, while the sperm density in the seminiferous
tubule lumen was similar to the stress group. The histopathological
images are shown in Fig. 4.
In this study, which was conducted to examine the changes that
stress can cause in the reproductive system in male rats and the
possible effects of Melatonin on this situation; To conrm whether
a chronic unpredictable stress pattern has occurred, serum
corticosterone levels were measured, and serum corticosterone levels
were found to be high, it is the primary glucocorticoid corticosteroid
FIGURE 3. Testicular tissue oxidative stress and cytokine levels. Values are Mean ± SEM and different letters (a, b, c) indicated represent statistical signicance between
groups (P<0.05)
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secreted from the adrenal gland in rats (Wistar albino) [24]. This
nding shows that the chronic stress model applied is effective.
Studies have shown that exogenous Melatonin causes an increase
in Melatonin levels in both serum and testes [1, 15]. In the study, it
was measured the serum Melatonin levels to reveal the presence of
orally administered Melatonin in the circulatory system, and it was
found to be signicantly higher. Due to the toxicity of DMSO, which
is used as a solvent for some substances, it is recommended not to
use more concentrated than 10%. However, in cases where it is used,
it is recommended to add a new group to the study as an additional
solvent group [20]. Because it was dissolved Melatonin in 10% DMSO
in the present study, it was added the solvent group to the study.
Furthermore, it was found that the related parameters were not
different from the control group.
Guvenc et al. [21] and Guo et al. [1] showed that sperm parameters
were negatively correlated with MDA levels and positively correlated
with antioxidant enzyme levels. This study shows a similar correlation
between oxidative stress parameters and spermatological parameters
among the Stress and Stress+Melatonin groups. In addition, in the
present study, it was determined that the use of Melatonin for three
weeks in the M group did not cause a change in the testicular oxidant–
antioxidant balance. Guo et al. [1] showed that Melatonin treatment
did not affect testicular oxidant–antioxidant capacity in healthy male
mice (BALB/c) (Mus musculus).
FIGURE 4. Seminiferous tubule histological images of Control (C), Stress (S) and Stress+Melatonin (SM) groups A: Histological structure of the seminiferous tubules
(ST) of the magnication, H&E × 100 µm B: Histological structure of the ST lumen of the magnication, H&E
× 20 µm
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In the present study was found that stress increased proinammatory
cytokine levels in serum and testicular tissue and decreased anti–
inammatory cytokine (IL–10) levels. Also, it was found that the use of
Melatonin caused a decrease in increased proinammatory cytokine
levels and an increase in decreased anti–inammatory cytokine levels
in the SM group. Similar ndings are seen in previous studies [1, 16].
A study in mice showed that chronic unpredictable stress causes
deterioration in spermatological parameters (sperm count, motility,
abnormal acrosome) [4]. Nirupama et al. [3] stated in their study that
most of the semen damage caused by stress is irreversibly affected.
However, Guo et al. [1] reported that exogenous Melatonin increased
sperm density, was ineffective on testicular weight and was curative
against testicular damage in restraint stress mice. In addition, the use
of Melatonin in testicular damage caused by different stimuli causes an
increase in sperm count, motility, and sperm viability and a decrease
in abnormal sperm [25]. In the current study, while stress caused
deterioration in sperm, Melatonin statistically improved the sperm
damage caused by stress in the SM group.
Chronic stress causes decreases FSH, LH, and Testosterone levels
through Gonadotropin–releasing hormone (GnRH [ 26 ], LH reduction
also contributes to the reduction of Testosterone levels [27, 28]. On
the other hand, glucocorticoid levels that increase with stress cause
apoptosis in Leydig cells and decrease Testosterone production
[27]. In this study, 8–week stress decreased rats' serum FSH, LH,
and Testosterone levels.
The effects of Melatonin on reproductive system hormones have
been studied for a long time, but its effects are still unclear. Wu
et al. [29] showed that rats that were using Melatonin inhibited
Testosterone release via cyclic adenosine monophosphate (cAMP) in
a dose–dependent manner by affecting Leydig cells, and the Melatonin
receptor antagonist luzindole eliminated this effect. The effect of
Melatonin is also variable in studies conducted on women. For example,
it has been observed that chronic Melatonin administration reduces
LH levels [30], and acute Melatonin use causes an increase in LH
levels in premenopausal women with regular cycles [31]. It has been
argued that LH release was higher in patients who received low–dose
Melatonin therapy for one year for sleep disorders than in healthy adults.
However, it is early to argue for a denitive role of Melatonin as a central
or peripheral modulator of the reproductive system [32]. It has been
reported that the levels of FSH, LH, and Testosterone in male rats
administered Melatonin decrease depending on the dose and time [33].
On the other hand, no correlation was found between low Testosterone
levels in people taking low doses of Melatonin [34]. In the present study,
it was observed that the use of Melatonin decreased serum FSH, LH,
and Testosterone levels in both healthy and stressed rats. It will likely
decrease reproductive hormones by showing a cumulative effect when
Melatonin and stress are applied together.
Paracrine/autocrine factors play important roles in
Spermatogenesis. Testicular cytokines and growth factors are
produced in many places in the testis, including Sertoli cells, Leydig
cells, Peritubular cells, spermatogonia, differentiated spermatogonia,
and even spermatozoa. It has been shown to affect the function
and secretion of Leydig and Sertoli cells [35]. Guo et al. [1] stated
that Melatonin has an antiapoptotic effect using the NFKB/i–NOS
pathway and an antioxidant effect using the NRF2–HO–1 pathway
in spermatological parameters that decrease in restraint–stressed
mice. According to Bahrami et al. [36], exogenous Melatonin has
an antioxidant effect by increasing total antioxidant capacity,
antioxidant enzyme levels such as GSH, decreasing MDA levels, which
is an indicator of lipid peroxidation levels, and an anti–inammatory
effect by reducing proinammatory cytokine levels such as IL–1β and
TNF–α, which has a healing effect on spermatological parameters
in reproductive damage. In this study, spermatological parameters
were not adversely affected despite the decreased hormone levels.
This situation can be explained by the fact that Spermatogenesis is a
complex process, and spermatological parameters are also affected
by non–hormonal mechanisms.
Stress also causes histological changes in testicular tissue. For
example, it is known that chronic stress causes deterioration in
testicular functions by affecting the tight junction proteins in the
blood testicular barrier structure [4]. Koksal et al. [37] showed that
Melatonin brought the histopathological changes closer to the control
in the ipsilateral and wholly normalized in the contralateral in rats
with testicular ischemia–reperfusion. A study examining Melatonin's
protective effect in acute testicular torsion found that Melatonin partially
corrected histological deterioration [38]. In the present study was found
that the morphological changes caused by chronic unpredictable stress
in rats were partially corrected by exogenous Melatonin.
CONCLUSION
In the current study, stress application causes decrease in
spermatological parameters by increasing serum corticosterone
levels, oxidative stress, inammatory cytokines, and decreasing
reproductive hormone levels. It was observed that the administration
of Melatonin in stress groups caused improvements in spermatological
parameters by improving the oxidant–antioxidant balance and
reducing inammatory cytokines. In conclusion, 10 mg·kg
-1
·day
-1
of
Melatonin for three weeks reduces serum glucocorticoid levels. It
stimulates antioxidant and anti–inammatory mechanisms, resulting
in improvements in sperm parameters. However, in pathophysiological
conditions such as stress, hormonal status should be considered
when used for longer than the specied dose and time.
ACKNOWLEDGEMENTS
This manuscript is derived from the PhD thesis of the rst author,
and some of the ndings were presented at the scientic meeting
(Turkish Society of Physiological Sciences, 46th Turkish Physiology
Congress, 8 – 10 Oct. 2021).
Conict of Interests
The authors of this study declare that there is no conict of interest
with the publication of this manuscript.
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