Effects of Crocin on The Pituitary-Gonadal Axis and Hypothalamic Kiss-1 Gene Expression in Female Wistar Rats
Materials and Methods
In this experimental study, 18 adult female Wistar rats were randomly divided into three
groups. Control group received normal saline and experimental groups received two different doses of crocin (100
and 200 mg/kg) every two days for 30 days. After the treatment period, blood samples were obtained from the
heart and centrifuged. Next, the serum levels of follicle-stimulating hormone (FSH) and luteinizing hormone (LH),
estrogen and progesterone hormones were measured by ELISA assay. The ovarian tissues were removed and fixed
for histological investigation. The hypothalamic
A significant reduction (P=0.038) in the number of atretic graafian follicles (0.5 ± 0.31) was observed in
rats treated with 200 mg/kg crocin. In addition, estrogen concentration in experimental groups (35.04 ± 0.85 and
36.18 ± 0.69 in crocin 100 and 200 mg/kg groups, respectively) compared to control group (38.35 ± 0.64) and
progesterone concentration in rats treated with crocin 200 mg/kg (2.06 ± 0.07) compared to control group (2.16
± 0.04), significantly decreased. Interestingly, relative expressions of
Crocin, at hypothalamic level, reduces Kiss-1 gene expression and it can prevent follicular atresia and reduce serum levels of estrogen and progesterone.
Hypothalamic-pituitary-gonadal axis (HPG axis) has
an important role in hormonal regulation of reproductive
system. Disruption of this axis can have unpleasant
consequences on fertility (1). Kisspeptin, also known as
metastin, is a hypothalamic peptide encoded by the
In rodents’ central nervous system, Kisspeptin expressing neurons were found in the anteroventral periventricular nucleus (AVPV) and arcuate nucleus (ARC) of the hypothalamus (5). Kisspeptin neurons send projections to gonadotropin- releasing hormone (GnRH) cell bodies, regulate the secretion of GnRH (6) and thereby control the release of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) (7). Based on its role in sex organ development and the HPG-axis, kisspeptin neurons dysfunction can lead to abnormal fetal development and infertility (8).
Medicinal plants like
Materials and Methods
All aspects of animal care complied with the ethical guidelines and technical requirements approved by the Institutional Animal Ethics Committee. In this study, after two-week adaptation period, 18 virgin adult female Wistar rats were maintained under standard laboratory conditions. Rats (160-180 g) were housed under controlled lighting (12 hours light and 12 hours dark) at 20 ± 2°C and had free access to food and water. Synchronization of estrus in rats was performed using estradiol valerate and progesterone. Estrous cycle was monitored by vaginal smears.
In this experimental study, 18 Rats were randomly divided into three groups as follows: control group received 2 ml normal saline, experimental group I received crocin 100 mg/kg body weight (BW) intraperitoneally (Pharmaceutical Research Center, BuAli Research Institute, Mashhad University of Medical Sciences, Mashhad, Iran) and experimental group II received crocin 200mg/kg BW intraperitoneally every two days for 30 days (22).
After the treatment period, animals in each group were anesthetized by ketamine/xylazine (k,80-100 mg/kg, X, 10-12.5 mg/kg) and blood samples were collected from their hearts. Blood samples were centrifuged for 10 minutes at 8000 rpm and the serum were separated and stored at -20°C. ELISA technique was used for evaluation of FSH, LH (commercial kits purchased from Pishtaz Teb Co., Iran), estrogen and progesterone hormones (commercial kits purchased from DRG Co., Germany).
The left ovary of animals were removed and fixed in 10% formalin solution. The specimens were processed through routine paraffin embedding method. Subsequently, 6 µm serial paraffin sections were stained with haematoxylin and eosin (H&E). The total number of sections was counted and the middle section of the ovary was determined. The follicles were counted in 5 sections per ovary which included the middle section and 4 sections from either side of the center. Ovarian follicle counting was performed (23) and atretic graafian follicles were identified (24) and counted using a light microscope (Olympus IX71, Japan).
Kiss-1 gene expression by quantitative
real-time polymerase chain reaction
Brains were removed immediately from the skull. The
hypothalami were dissected and frozen at -80°C. These
tissues were thoroughly homogenized. Total RNA was
extracted using RNeasy Mini Kit (Qiagen, Germany) and
RNA concentration was determined by Nano Drop ND-
1000. cDNA synthesis was performed using 2 µg of total
RNA and QuantiTect Reverse Transcription Kit (Qiagen,
Germany). The quality of synthesized cDNA was assessed
by polymerase chain reaction (PCR) using
|Primer||Primer sequencing (5ˊ-3ˊ)||Product length (bp)||Annealing temperature (°C)|
Real-time PCR was performed in a thermal cycler Rotor
gene 6000 (Corbett, AUS). The PCR mixture for each
reaction contained 5 µl SYBR premix Ex Taq II, 0.5 µl
of each primer (5 pmol/µl) and 50 ng cDNA adjusted to
a final volume of 10 µl using DEPC water. All reactions
were carried out in triplicate. The real-time PCR protocol
included 5 minutes at 95°C followed by 40 repetitive cycles
for 10 seconds at 95°C, 30 seconds at 60°C and 61°C
The results were statistically analyzed using SPSS 19 software. Mean ± SD was calculated for each parameter and differences among means were evaluated by ANOVA followed by the Tukey post-hoc test using the Excel computer-based program. P<0.05 were considered statistically significant.
There was no significant differences in FSH hormone levels
between experimental groups (0.33 ± 0.042 for crocin
100 mg/kg, P=0.158 and 0.34 ± 0.073 for crocin 200 mg/
kg, P=0.302) and control group (0.36 ± 0.008). In addition,
administration of crocin 100 and 200 mg/kg doses had no
effect on LH levels in female rats ((0.66 ± 0.026, P=0.120)
and (0.67 ± 0.032), P=0.350, respectivly) compared to control
groups (0.69 ± 0.019) (Fig .1,). However, both doses of
crocin significantly decreased serum estrogen levels (35.04
± 0.85 for crocin 100 mg/kg, P=0.000) and (36.18 ± 0.69
for crocin 200 mg/kg, P=0.000) compared to the control
group (38.35 ± 0.64) (Fig .2,). Also, progesterone concentrations
significantly reduced in rats received 200 mg/kg
crocin (2.06 ± 0.07, P=0.009) compared to control group
(2.16 ± 0.04) (Fig .1,). The expression of
|- Comparison of serum estrogen level among the experimental and control groups. Significant differences were observed between groups treated with Crocin and control group (P<0.05).|
|- The ovarian tissue. Photomicrographs of ovary tissue (6-µm thick sections were stained with H&E; X100) in A. The control animals and B. Rats treated with 200 mg/kg crocin. AF; Atretic graafian follicle (scale bar: 100 µm).|
||P value compared to control group|
|Control||1 ± 0||-|
|Crocin (100 mg/kg)||0.00053 ± 0.0005a||0.000|
|Crocin (200 mg/kg)||0.0011 ± 0.0007a||0.000|
All data were presented as mean ± SD.
a; Indicates a significant difference between experimental groups and control group (P<0.001).
|Variable||Control||100 mg/kg/72 hours||200 mg/kg/72 hours||P value compared to control group|
|Primordial follicles||8.5 ± 1.169||5.8 ± 0.98||5.8 ± 1.17||0.0520.056|
|Primary follicles||2.17 ± 1.169||3.17 ± 0.408||1.33 ± 0.516||0.0970.183|
|Growing follicles||1.67 ± 0.516||1.17 ± 0.408||1.17 ± 0.408||0.1630.163|
|Graafian follicles||1.5 ± 0.837||1.17 ± 0.753||1.83 ± 0.753||0.7450.745|
|Atretic Graafian follicles||1.33 ± 0.448||1.33 ± 0.448||0.5 ± 0.31a||10.038|
|Corpora lutea||2.5 ± 0.548||4 ± 1.549||4 ± 1.265||0.1090.109|
All data were presented as mean ± SD.
a; Indicates a significant difference between Crocin 200 mg/kg-treated group and control group (P<0.05).
Along with other hypothalamic factors, the
Considering the fact that estrogen is secreted by follicular
cells in the ovary, this may explain a role for crocin
at the ovarian level. In this regard, previous studies have
shown that carotenoids reduce the activity of cytochrome
p450, thus inhibiting the transformation of cholesterol to
pregnenolone, and consequently reducing the amount of
estrogen. This effect of carotenoids is believed to be mediated
by reduced expression of the
Considering the reduction in estrogen, an increase
Also, differential action of estrogen on
Following hormonal evaluation, we assessed histological
sections for any alteration in folliculogenesis. The results
revealed no significant changes in the mean value
of the number of different follicles between the control
and treated groups except for a reduction in the number of
atretic graafian follicles which were significantly reduced
in crocin 200 mg/kg-treated group. This observation may
be related to the anti-apoptotic effect of carotenoids, like
crocin. Carotenoids up-regulate the expression of
Another process involved in the generation of atretic follicles is excessive production of reactive oxygen species (ROS). Assimopoulou et al. (13) showed that crocin has a marked radical-scavenging activity. In this regard, Soeda et al. (35) reported that crocin inhibits oxidative stress- induced cell death via a glutathione (GSH)-dependent mechanism. Also, Hosseinzadeh et al. (36) showed that crocin decreases malondialdehyde (MDA) generation. The role of crocin as an anti-apoptotic agent is well established in different systems (37, 38).
As ovarian follicles synthesize estrogen, one may expect that a decrease in the number of atretic follicles may result in an increase in estrogen production, which is contrary to results showing a reduction in estrogen levels. This effect might be due to a reduction in aromatase activity induced by crocin. The reduction in estrogen might be possibly due to crocin effect on the ovary rather than on the hypothalamus, as crocin had no effects on FSH and LH levels.
Based on the literature, crocin can induce the expression
of genes like
Our results revealed that at the hypothalamic level,
This work was financially supported by Science and Research Branch, Islamic Azad University, Tehran, Iran. There is no conflict of interest in this article.
K.P., M.H.S., N.H.R.; Contributed to conception and design. D.Z.; Performed data collection and evaluation, drafting and statistical analysis. All authors performed editing and approving the final version of this paper for submission, also participated in the finalization of the manuscript and approved the final draft.