Peptides 163 (2023) 170976

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Peptides
journal homepage: www.elsevier.com/locate/peptides

Investigation of irisin’s role in pubertal onset physiology in female rats

Esra Kutlu a, *, lker Tolga Ozgen b, Huri Bulut c, Abdurrahim Kocyigit d, Savas Ustunova e,
Onder Hüseyinbas f, Emel Torun g, Yasar Cesur b
a
Health Sciences University Istanbul Umraniye Training and Research Hospital, Department of Pediatrics, Division of Pediatric Endocrinology, Umraniye, Istanbul,
Turkey
b
Bezmialem Vakif University, Faculty of Medicine, Department of Pediatrics, Division of Pediatric Endocrinology, Fatih, Istanbul, Turkey
c
Istinye University, Faculty of Medicine, Department of Biochemistry, Zeytinburnu, Istanbul, Turkey
d
Bezmialem Vakif University, Faculty of Medicine, Department of Biochemistry, Fatih, Istanbul, Turkey
e
Bezmialem Vakif University, Faculty of Medicine, Department of Physiology, Fatih, Istanbul, Turkey
f
Bezmialem Vakif University, Faculty of Medicine, Animal Research Laboratory, Fatih, Istanbul, Turkey
g
Bezmialem Vakif University, Faculty of Medicine, Department of Pediatrics, Fatih, Istanbul, Turkey

A R T I C L E I N F O A B S T R A C T

Keywords: Objective: The timing of pubertal development is closely related to metabolic status and energy reserves. It is
Puberty thought that irisin, which is involved in the regulation of energy metabolism and is shown to be present in the
HPG axis hypothalamo-pituitary-gonadal (HPG) axis, may play a role in this process. In our study, we aimed to investigate
İrisin
the effect of irisin administration on pubertal development and HPG axis in rats.
GnRH
KNDy
Design-methods: 36 female rats were included in the study were divided into 3 groups: 100 ng/kg/day irisin
treatment group (irisin-100), 50 ng/kg/day irisin treatment group (irisin-50), and control group. On the 38th
day, serum samples were taken to determine levels of luteinizing hormone (LH) and follicle stimulating hormone
(FSH), estradiol and irisin. Brain hypothalamus samples were taken to determine levels of pulsatile
gonadotropin-releasing hormone (GnRH), kisspeptin, neurokinin-B, dynorphin (Dyn), and makorin ring finger
protein-3 (MKRN3).
Results: Vaginal opening and estrus were seen firstly in the irisin-100 group. At the end of the study, the highest
rate of vaginal patency was found in the irisin-100 group. Hypothalamic protein expression levels of GnRH, NKB
and Kiss1 in homogenates; serum FSH, LH, and estradiol levels were the highest in the irisin-100 group, followed
by the irisin-50 and control groups, respectively. Ovarian sizes were significantly greater in the irisin-100 group
compared to the other groups. The hypothalamic protein expression levels of MKRN3 and Dyn were the lowest in
the irisin-100 group.
Conclusions: In this experimental study, irisin triggered the onset of puberty in a dose-dependent manner. Irisin
administration caused the excitatory system to dominate in the hypothalamic GnRH pulse generator.

1. Introduction
Puberty is a period of somatic and behavioral change in which sexual
maturation and reproductive capacity are gained. Puberty begins with
the activation of the hypothalamo-pituitary-gonadal (HPG) axis. While
the HPG is active in the intrauterine and mini puberty (between the first
week of life and 6–9 months) periods, it remains suppressed afterwards
until the onset of puberty [1]. Physiologically, it is reactivated between
the ages of 8–13 in girls and 9–14 in boys. The HPG axis regulates the
secretion of pulsatile gonadotropin-releasing hormone (GnRH), lutei­
nizing hormone (LH) and follicle stimulating hormone (FSH), which
play a key role in the control of gonadal maturation and functions [2].
The timing of pubertal onset is determined by the neuroendocrine
network as well as metabolic and environmental factors. Although
various hypotheses have been proposed, the exact mechanism under­
lying the timing of pubertal onset is not fully understood. It was stated
by Baker in 1985 that a critical body weight is required for HPG axis
activation and the ratio of lean mass to fat mass could be important in
this regard [3]. When a critical fat/muscle mass is gained, metabolic
signals transmit this information to the hypothalamic neuronal network.
GnRH release is regulated by changing the balance of excitatory and
inhibitory factors in the GnRH pulse generator and upstream pathways.

* Correspondence to: Health Sciences University Istanbul Umraniye Training and Research Hospital, Umraniye, Istanbul, Turkey.
E-mail address: esrakutlu07@gmail.com (E. Kutlu).

https://doi.org/10.1016/j.peptides.2023.170976
Received 29 September 2022; Received in revised form 9 January 2023; Accepted 13 February 2023
Available online 14 February 2023
0196-9781/© 2023 Elsevier Inc. All rights reserved.
E. Kutlu et al.
Gamma aminobutyric acid (GABA), glutamate, kisspeptin (KISS1),
neuropeptide-Y, neurokinin-b (NKB), dynorphin (Dyn), makorin ring
finger protein-3 (MKRN3), MicroRNAs (MiRNAs), endorphins, opioids,
and melatonin seem to contribute to the regulation of these pathways [2,
4].
A group of neurons which co-express KISS1, NKB, and Dyn located in
the arcuate/infundibular nucleus of the hypothalamus have been named
as "KNDy" [5]. In the GnRH pulse generator, KISS1 and NKB have an
excitatory effect, while Dyn has an inhibitory effect [6]. MKRN3 is
another inhibitor of this generator [7]. As a result, with the increase of
excitatory stimuli and decrease of inhibitory stimuli, the release of
pulsatile GnRH increases, and pubertal activation begins. However,
what triggers the change balance of the excitatory/inhibitory system is
not clear.
Metabolic control and energy metabolism is another important factor
affecting pubertal onset, especially in girls. Changes occur in body
composition and insulin sensitivity in the peripubertal period. Obesity
and overnutrition are associated with an earlier onset of puberty. On the
other hand, malnutrition and intense physical activity are reported to
delay the onset of puberty [8,9]. The role of various metabolic signals
such as leptin, insulin, ghrelin, preadipocyte factor 1 (Pref-1), and
ceramides on the timing of puberty onset have been previously inves­
tigated and those studies have shed some light on this issue [10,11].
However, the exact mechanisms and mediators of metabolic control of
puberty have not yet been fully elucidated.
Irisin is an adipomyokine involved in the modulation of metabolic
processes. Irisin is encoded by the fibronectin type-III domain-containing
protein-5 (FNDC5) gene through peroxisome proliferator-activated re­
ceptor-γ coactivator 1α (PGC-1α) activity, especially in skeletal muscles
and subcutaneous and visceral adipose tissues. Irisin plays a role in
browning of white adipose tissues and thermogenesis. Irisin has been
shown to be present in testicles, ovaries, uterus, central nervous system
(cortex, hippocampus, putamen, hypothalamus, pituitary and cere­
bellum), peripheral nervous system, and adrenal gland tissues [12].
Irisin has been reported to play a role in the reproductive system at both
central and gonadal levels [13–15]. It has been reported that hypotha­
lamic FNDC5 expression increases at puberty in mice, thus irisin seems
to have important effects in the CNS as well [13]. In a longitudinal study
by Reinehr et al. [16] in obese children, irisin levels were reported to
increase significantly with the onset of puberty.
In the light of all this information, irisin may have a role in the onset
of puberty. However, there are limited number of studies which inves­
tigated the relation of irisin with pubertal onset. Moreover, in our
knowledge there is no study about the effect of irisin on upstream
pathways of the GnRH pulse generator. In our earlier study, we exam­
ined the relationship between serum irisin levels and central precocious
puberty (CPP) and premature thelarche (PT) [17]. In that study, irisin
levels were higher in subjects with CPP compared with healthy controls
and subjects with PT. We also detected significant associations between
irisin levels and pubertal activation indicators in that study. However, it
is not yet known whether irisin triggers the initiation of puberty or it
solely accompanies the increased muscle and fat mass during the pu­
berty process. In the present study, we aimed to investigate the role of
irisin in pubertal physiology and its effect on the hypothalamic pulse
generator and upstream pathways by examining the effects of recom­
binant irisin on pubertal development and the protein expression of
KNDy and MKRN3 in rats.
2. Materials and methods
2.1. Animals
Bezmialem Vakıf University Clinical Research Ethics Committee
approved the study protocol (1348-2019/135, 2020/51). In the study,
one-day-old 36 Wistar Albino female rats were obtained from Bezmia­
lem Vakıf University Experimental Animals Laboratory. The rats were
2
Peptides 163 (2023) 170976
weaned at day 21 postnatal (PN) and housed in standard polycarbonate
cages. During the study, animals were kept in a controlled environment
at a stable room temperature (21 ± 1 ◦ C) with 55 ± 5 % humidity and an
automatically controlled 12-hour light (from 07.00 a.m. to 07.00 p.m.)
and 12-hour dark cycle. Pelleted food and tap water were provided. The
same environmental conditions and diet were applied to all rats.
2.2. Irisin injections
The rats were divided into three groups: 100 ng/kg/day irisin
treatment group (irisin 100) (n = 12), 50 ng/kg/day irisin treatment
group (irisin 50) (n = 12) and a control group which received saline
injection (n = 12). Intraperitoneal (IP) recombinant irisin (SRP6542,
Sigma-Aldrich, USA) (0.1 g/L in deionized water) was administered in
the irisin 50 and irisin 100 groups and an equal volume of IP saline was
administered in the control group between 08.00 and 09.00 am for 15
days beginning from the 15th day PN. The doses of irisin were preferred
according to the dose used in a previous study [15]. The flow chart of the
study is shown in Fig. 1.
2.3. Daily assessments
Daily weight measurement, vaginal opening, and first estrus exam­
ination were performed in all rats from the 15th day PN onwards. The
mean weight of all rats on the 15th day PN was 20 ± 2 g. To determine
the onset of puberty in all rats, external indices of vaginal opening were
evaluated. During the follow-up, development of vaginal opening and
onset of estrus were recorded daily until the 38th day. Vaginal lavages
were taken.
2.4. Termination of the experiment and collection of tissues
On the 38th day PN, all rats were decapitated after their vaginal
patency and weight were recorded. Blood samples were collected from
the rats and centrifuged (4000 g for 15 min at +4 ◦ C) and then stored at
− 80 ◦ C until the analyzes were performed. Brain hypothalamus samples
were obtained to determine protein expression levels of GnRH, Kiss1,
NKB, Dyn, and MKRN3. The tissue samples were stored at − 80 ◦ C until
analysis. Hypothalamus tissues were fixed in 10 % formaldehyde solu­
tion for immunohistochemical evaluation. In addition, the sizes of the
ovaries and uterus of the groups were evaluated macroscopically.
2.5. Hormone measurements
In the serum samples, FSH, LH, estradiol, and irisin levels were
measured using commercially available kits (Rat FSH Elisa kit, E-EL-
R0391, Elabscience, USA; Rat LH elisa kit, E-EL-R0026, Elabscience,
USA; Rat E2 Elisa kit, E-EL-0152, Elabscience, USA; Rat Irisin Elisa kit, E-
EL-R1104, Elabscience, USA) with the ELISA method. Optical density
was measured spectrophotometrically at 450 nm ± 2 nm wavelength
(Thermo Scientific Microplate Reader, USA). Detection ranges of FSH,
LH, estradiol, and irisin kits were 3.13–200 ng/mL, 1.56–100 mIU/mL,
1.56–100 pg/mL, and 62.50–4000 pg/mL, respectively.
2.6. Homogenization of the tissues
Equal volumes of hypothalamus tissues were taken into dry tubes
and homogenized in a RIPA buffer (MP Biomedicals FastPrep-24 5 G
Homogenizer, USA; 30 m/s for 5 min). The total protein quantities in
the samples were measured with Bradford protein assay kit (23200,
Thermofisher scientific, USA) in a 595 nm wavelength plate reader
(Thermo Scientific Multiskan FC, 2011-06, USA). As standard, 1 mg of
bovine serum albumin was used.
E. Kutlu et al. Peptides 163 (2023) 170976

Fig. 1. The flow chart of the experimental studies.

2.7. Western blot analysis

Protein expression levels of GnRH (ab112295, Abcam, UK), Kiss1

(ab19028, Abcam, UK), NKB (GTX16441, GeneTex Inc., USA), Dyn
(H00005173, Novus Biotech, UK), and MKRN3 (ABIN563435,
Antibodies-online GmbH, Germany) in homogenates were determined
by western blot analysis. GAPDH (Cell signaling, 14C10, USA) was used
as housekeeping protein. Primary antibodies of GAPDH, GnRH, Kiss1,
NKB, Dyn, and MKRN3 were prepared in 2.5 % skim milk at the ratios of
1/2000, 1/500, 1/250, 1/250, 1/200 and 1/250, respectively. The
secondary antibody (Santa Cruz Biotechnology, Inc., Santa Cruz Ca USA)
was prepared at a ratio of 1/1000. The formed bands of antibodies were
viewed and evaluated on the Vilbert Lourmart Fusion Fx5 device. Fig. 2. Difference between the groups in terms of body weight.

2.8. Statistical analysis Table 1

Number and rates of vaginal openings seen by days.
Statistical analyzes were performed with Instat Statistical Package 34. 35. day 36. day 37. day 38. day
Program (GraphPad Prism Version-6 Software Program San Diego, CA, day
USA). The Shapiro–Wilk test was used to test the normality of the data. Control group 0 2/12 5/12 6/12 7/12
As the data showed normal distribution, study groups were compared (16.6 %) (41.6 %) (50 %) (58.3 %)
using one-way ANOVA and post-hoc comparisons were performed with Irisin 50 0 1/12 5/12 7/12 8/12
Bonferroni test. The group means were expressed as mean ± standard group (83 %) (41.6 %) (58.3 %) (66.6 %)
(50 ng/kg/
deviation (SD). Statistical significance was considered where p was
day)
lower than 0.05. Irisin 100 1/12 3/12 9/12 10/12 10/12
group (83 %) (25 %) (75 %) (83.3 %) (83.3 %)
3. Results (100 ng/
kg/day)

The mean body weight of the groups were similar in the beginning
and at the end of the study (Fig. 2). Vaginal patency and estrus were
firstly seen in the irisin 100 group. At the end of the study, the highest
rate of vaginal patency was found in the irisin 100 group (83.3 %),
followed by the irisin 50 (66.6 %), and control groups (58.3 %). The
number and percentages of rats with vaginal opening by days are given
in Table 1 and Fig. 3.
Serum FSH, LH, estradiol and irisin levels were significantly higher
both in Irisin 50 and Irisin 100 groups than that in the control group
(p < 0.01 for FSH, p < 0.001 for LH, estradiol, and irisin). These were
also higher in the Irisin 100 group than that in the Irisin 50 group
(p < 0.01 for FSH, LH and estradiol, p < 0.001 for irisin). The highest
mean ovarian size was measured in the irisin 100 group (p = 0.019). The
uterine size was similar in all groups (Fig. 1). Comparison of laboratory
variables between the study groups is listed in Table 2.
Irisin administration led to a dose-dependent increase in GnRH levels

3
E. Kutlu et al. Peptides 163 (2023) 170976

adipose tissue and skeletal muscle stimulates the browning of white

adipose tissue, resulting in an increase in energy consumption, oxygen
utilization in adipocytes, and thermogenesis. Studies have shown that
irisin level is positively correlated with body mass index (BMI),
anthropometric parameters, fat mass, muscle mass, and physical activity
[17,20].
Administration of irisin seems to affect both body weight and func­
tions of the reproductive system. Bastu et al. [21] reported that seden­
tary mice which received irisin for around two weeks after the age of 20
weeks had significantly lower weight gain compared with the sedentary
control group. Both irisin administration and exercise were associated
Fig. 3. Vaginal opening by days. with higher numbers of primary and secondary follicles per ovary
compared to the control group [21]. The authors suggested that the
metabolic effects of irisin improved reproductive functions. However,
Table 2 the findings of the present study suggest that irisin may improve ovarian
Comparison of clinical and laboratory results between groups. functions by its effects on the HPG axis even regardless of the change in
Control Irisin 50 Group Irisin 100 Group p weight.
Group n:12 (50 ng/kg/day) (100 ng/kg/day) Irisin level has been reported to be higher in obese children
n:12 n:12
compared to normal-weight children [16]. The authors also reported
FSH (ng/ 69.4 ± 38 114.2 ± 38 164.1 ± 23 < 0.001 that the increase in irisin level was significantly associated with entry
mL) into puberty independent of the change in BMI [16]. Similarly, in our
LH (mIU/ 43.6 ± 19 72.9 ± 18 96.2 ± 14 < 0.001
mL)
previous study involving non-obese girls, irisin levels were significantly
E2 (pg/ 32.2 ± 14 54.5 ± 14 72.1 ± 10 < 0.001 higher in girls with CPP and in girls with PT (who are considered as
mL) having partial activation of the HPG axis) compared with the control
Irisin (pg/ 2010 ± 514 2833 ± 426 3629 ± 486 < 0.001 group who did not reach puberty. We reported that baseline FSH-LH and
mL)
peak LH were positively correlated with pubertal activation indicators
Ovary size 4.92 ± 0.9 4.83 ± 0.71 5.75 ± 0.86 0.019
(mm) such as bone age and ovarian size. The strongest correlation was seen
Uterus 24.25 ± 5.3 26.5 ± 6.2 28.08 ± 2.7 0.186 with peak LH [17]. Poretsky et al. [14] reported that irisin administra­
size tion stimulated LH production in the murine pituitary cells and also E2
(mm) production in ovarian granulosa cells. Wahab et al. [13] reported that
irisin and estrogen-related receptor alpha (ERRα) were co-localized in
(obtained from the hypothalamus tissue samples). The irisin 100 group the primate hypothalamus. They also reported that
had a higher mean GnRH level (1.42 ± 0.24) compared with the other irisin-immunoreactive fibers and GnRH-immunoreactive cells interacted
two groups. The irisin 50 group tended to have a higher mean GnRH and FNDC5/irisin expression which increased with the onset of puberty
level (0.91 ± 0.05) than the control group (0.61 ± 0.08), but the dif­ in both male and female monkeys. In addition, they observed a stimu­
ference was not statistically significant (Fig. 4). The mean levels of NKB latory effect of irisin on GnRH expression and release in mouse hypo­
and Kiss1, were higher in the irisin 100 group (NKB: 3.58 ± 0.35; Kiss1: thalamic cells [13]. In line with these findings, we observed in the
2.19 ± 0.54) compared with the irisin 50 and control groups. The mean present study that different doses of irisin triggered the onset of puberty
NKB and Kiss1 levels were higher in the irisin 50 group (NKB: 2.39 and predated the occurrence of first estrus. These effects were more
± 0.63; Kiss1: 1.84 ± 0.25) than the control group (NKB: 0.52 ± 0.25; pronounced with the higher irisin dose used in the present study. In
KISS1: 0.60 ± 0.46). The mean levels of MKRN3 and Dyn, were higher in addition, the hypothalamic GnRH level; serum FSH, LH, and estradiol
the control group (MKRN3: 2.03 ± 0.52; Dyn: 1.60 ± 0.39) compared levels; and ovarian dimensions were positively correlated with the irisin
with the irisin 100 and irisin 50 groups. The irisin 50 group had higher levels.
mean MKRN3 and Dyn levels (MKRN3: 1.09 ± 0.48; Dyn: 0.83 ± 0.46) In another recent study, chronic irisin exposure (10 weeks) was re­
compared with the irisin 100 group ((MKRN3: 0.42 ± 0.43; Dyn: 0.36 ported to delay the onset of puberty, decrease serum FSH levels, and
± 0.40) (Fig. 4). increase LH and E2 levels in female rats [22]. It is known that the HPG
axis is affected by long-term or high-intensity exercise, causing delay in
4. Discussion puberty, amenorrhea, and impairment in reproductive functions in girls
[23]. In the present study, we aimed to imitate the physiological acti­
Adolescence is a highly complex physiological process that results in vation of the HPG axis by increasing irisin level for a short duration.
the acquisition of psychosexual maturity and reproductive capacity. The most important step for pubertal activation is the activation of
Many factors, especially genetic, metabolic, and environmental condi­ the HPG axis and the initiation of pulsatile GnRH release. Irisin has been
tions, determine the timing of pubertal development. The HPG axis is shown to increase hypothalamic GnRH release. However, it is not yet
suppressed until the prepubertal period. Afterwards it is reactivated due known how and through which pathways irisin causes an increase in
to the increase in pulsatile GnRH release. The neuronal network called hypothalamic GnRH release. In the present study, to the best of our
"GnRH pulse generator" is controlled by stimulatory and inhibitory knowledge, the effect of irisin on the hypothalamic pulse generator and
signals originating from the hypothalamic arcuate and infundibular upstream pathways was investigated for the first time.
regions. The important role of KISS1 in activating the HPG axis was first
It is postulated that metabolic status and energy metabolism play an discovered in cases of hypogonadotropic hypogonadism which is due to
important role in the physiological activation of this network. Irisin may inactivating mutations in the kisspeptin receptor and KISS1 gene [24].
be a candidate signal molecule that transmits information about body Similarly, some case series suggested that hypogonadotropic hypo­
weight (fat/muscle mass) to the HPG axis [14]. Irisin level measured in gonadism developed due to inactivating mutations in TAC3 and TAC3R
the CSF are proportional to that in systemic circulation (~ 1/20–25). genes encoding NKB and its receptor [25]. Navarro et al. [26] reported
Irisin level in the CSF is higher than other adipokines such as leptin, that the onset of puberty and first vaginal opening were delayed in rats
adiponectin, and resistin [18,19]. In particular, irisin secreted from which were administered an NKB receptor antagonist. Goodman et al.
[27] reported that Dyn, which is encoded by the Prodynorphin gene, was

4
E. Kutlu et al.
Fig. 4. Comparison of laboratory results of hypothalamus tissue samples. *: Significance relative to control, +: Significance between irisin 100 and irisin 50. *, +:
p < 0,05; **, ++: p < 0,01; ***, +++: p < 0001.
secreted from Kiss1/NKB neurons and inhibited GnRH secretion. The
effect of Dyn on GnRH secretion was supported by the fact that
administration of a Dyn receptor antagonist led to advancement of pu­
berty onset in female rats [28]. This co-ordinated neuron cluster was
named "KNDy" neurons by Cheng et al. in 2010 [6]. The MKRN3 gene is
reported to be involved in protein ubiquitination and cell signaling. The
inactivating mutations in the MKRN3 gene led to CPP. In animal models,
MKRN3 activity is reported to decrease and the suppressor effect of
MKRN3 on the Kiss1 and TAC3 genes diminish during the onset of pu­
berty [29]. In summary, the increase of excitatory factors such as KISS1
and NKB and the decrease of inhibitory factors such as Dyn and MKRN3
lead to activation of the GnRH pulse generator, increase in pulsatile
GnRH release, and increment in the levels of gonadotropins (FSH, LH)
and sex steroids. Consequently, pubertal development begins.
In the present study, we observed that irisin administration led to a
dose-dependent increase in NKB and Kiss1 levels (which act as GnRH
stimulators), and a dose-dependent decrease in MKRN3 and Dyn levels
(which act as GnRH inhibitors). These findings suggest that the excit­
atory/inhibitory stimuli, which are balanced in the HPG axis in the
prepubertal period, shift to an excitatory state after short term
5
Peptides 163 (2023) 170976
administration of irisin. Its effect on both the excitatory and inhibitory
systems suggests that irisin may be an important trigger for pubertal
activation. In addition, the lack of difference in weight between the
groups throughout the study, suggests that the effects of irisin admin­
istration on puberty onset are not related with its effects on the increase
in body weight.
Among the limitations of the present study is the fact that we
measured only body weight and did not examine body composition in
detail. However, we think that body composition might not have a sig­
nificant effect on our study variables because of the similar weight of the
groups in the beginning and at the end of the study. The second limi­
tation was regarding the widespread and diverse effects of irisin on
multiple stimulatory and inhibitory pathways. It has been suggested that
some of the changes observed may be affected by irisin indirectly.
However, we could not exactly demonstrate in the present study, which
molecules or system were stimulated or suppressed. There is need for
further studies in this regard.
In summary, this experimental study in rats revealed that the
administration of irisin triggered puberty in a dose-dependent manner.
To the best of our knowledge, the mechanisms of action of irisin on the
E. Kutlu et al.
hypothalamic GnRH pulse generator and upstream pathways (KNDy,
MKRN 3) were examined for the first time. Our findings may point out to
the fact that irisin has an important role in transmitting the metabolic
signals necessary for pubertal activation to the hypothalamic neuronal
network and also in the regulation of GnRH release. However, more
detailed molecular and functional studies are needed to further char­
acterize the mechanisms of the effect of irisin on the neuroendocrine
system, in particular on the human HPG axis.
CRediT authorship contribution statement
EK: Literature search, study design, data collection, data interpre­
tation, Writing – original draft, Writing – review & editing, Methodol­
ogy, Project administration, Conceptualization. lTO: Study design,
Project administration, Methodology, Resources, Supervision, Valida­
tion, Writing – review & editing. HB: Data collection, data analysis, data
interpretation, Writing – original draft, Writing – review & editing,
Visualization, Investigation. AK: Resources, Software, Validation,
Funding acquisition. OH: Data collection, data interpretation, Method­
ology, Resources. SU: Data analysis, data interpretation, Writing –
original draft, Writing – review & editing, Visualization. ET: Literature
search, data collection. YC: lnvestigation, Funding acquisition, Writing –
review & editing.
Funding
This study is supported by Bezmialem Vakif University Scientific
Research Projects Foundation (Project number: 12.2017/36).
Disclosure Statement
The authors have nothing to disclose.
Data Availability
Data will be made available on request.
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