Document Type : Original Article
Authors
1 Department of Animal Sciences, College of Agriculture, Shiraz University, Shiraz, Iran
2 Stem Cell Technology Research Center, Shiraz University of Medical Sciences, Shiraz, Iran
3 Department of Basic Sciences, School of Veterinary Medicine, Shiraz University, Shiraz, Iran
4 4Department of Medical Biotechnology, School of Advanced Medical Sciences and Technology, Shiraz University of Medical Sciences, Shiraz, Iran;5Institute for Cancer Research, Shiraz University of Medical Sciences, Shiraz
5 6Cardiovascular Research Center, Shiraz University of Medical Sciences, Shiraz, Iran
6 7Comparative and Experimental Medicine Center, Shiraz University of Medical Sciences, Shiraz, Iran
Abstract
Keywords
Polycystic ovarian syndrome (PCOS) as an complex
endocrine disorder in women is accompanied with ovarian
dysfunction, metabolic disorders (e.g., obesity), and
a myriad of causes, including genetic abnormalities, fetal
epigenetic alterations, maternal or postpubertal hormonal
imbalances, lifestyle, and environmental factors have been
explained (
Common neuroendocrine disorder of PCOS, increased
frequency and amplitude pulses of gonadotropin releasing
hormone (GnRH) (
On the other hand, some evidences show that PCOS
may be originated from dysfunctions in regulating neuronal
circuits of negative feedback of steroids to hypothalamus-
pituitary-gonads (HPG) axis (
All experimental procedures on rats were performed based on the instructions of Animal Care Committee of Shiraz University. The experimental procedure had been approved by Chancellor of Research Committee of the Shiraz University.
In the experimental study, 30 female Sprague-Dawley rats
were purchased from and kept in the Center of Comparative
and Experimental Medicine, Shiraz University of Medical
Sciences. The rats were housed in standard condition of
laboratory animal center (22 ± 1°C temperature) and food
and water were available ad libitum. Twelve nulliparous (38
days-old, 177 ± 20 g) and 12 uniparous (80 days-old, 226
± 20 g) rats were randomly allocated into two PCOS and
control normal sub-groups (n=6). PCOS was induced using
constant light (
Six remained nulliparous rats were used as the ovariectomized control group for real-time polymerase chain reaction (PCR). The ovariectomy was performed through a ventral midline incision after anesthetizing with an intraperitoneal injection of xylazine (7 mg/kg, Alfazyne, Netherlands) and ketamine (100 mg/ kg, Netherlands). Brain tissue sampling in this group was done after two weeks.
For sampling, the rats were euthanized with ether and blood was sampled in tubes without anticoagulants by cardiac puncture. Serum was collected by centrifuging 2000 rpm for 15 minutes and then stored at -80°C until analysis.
Brain was dissected out from skull and DMH was sampled
(
Then ovaries were removed through ventral midline incision
and kept in 10% buffer formalin solution. Ovaries
were dehydrated by graded concentrations ethanol and xylene
and then were embedded in paraffin. Serial sectioning
was performed at thicknesses of 10 µm. Sections were
deparaffinized in 60°C. In room temperature, sections were
rehydrated in xylene and graded concentrations of ethanol.
Samples were stained with hematoxylin and eosin (H&E)
stain. Follicle types in ovarian sections were defined (
Serum concentration of testosterone with 0.2 nmol/L sensitivity (catalog# RK-61M, Institut des Isotopes Ltd, Hungary) was measured by radioimmunoassay (RIA) technique. In addition, serum concentrations of follicle stimulating hormone with 0.09 mIU/mL sensitivity (catalog# RF01N, Gyeonggi-do, South Korea) and luteinizing hormone with 0.22 mIU/mL sensitivity (catalog# RF03N, Gyeonggi-do, South Korea) were determined using immunoradiometric assay (IRMA) technique.
Designed primers for
Gene | Primer sequencing (5ˊ-3ˊ) | Amplicon length (bp) |
---|---|---|
F: AAGACACTGGCTGGTTTG | 192 | |
R: TTGAAGGACTGGCTGGAG | ||
F: CCACACTTTCTACAATGAGC | 169 | |
R: ATACAGGGACAACACAGC | ||
For quantitative assessment and evaluation of the relative
mRNA expression of
The normality of data from hormone measurements and
the
The microscopic evaluation of ovaries in the PCOS
groups showed formation of cystic follicles (
Alterations of histological charecters of ovaries in the female nulliparous and the primiparous rats after the exposition to continuous light during 90 days. The control groups show normal ovarian feature with Several corpus luteum (white stars) and normal tertiarry follicles (arrows). The polycystic ovary syndrome (PCOS)-induced groups showed considerably distended and cystic tertiary follicles [black stars, hematoxilin and eosin staining (H&E)]. A. Nulliparous control, B. Nulliparous PCOS, C. Uniparous control, and D. Uniparous PCOS.
Alterations of tertiarry follicles features in the female nulliparous and the primiparous rats after continuous light exposure during 90 days. Ovary of the control rat with normal tertiary follicles (white stars). Oocytes and corona radiata are absent in the polycystic ovary syndrome (PCOS)-induced groups and atretic follicles (black stars) are more observable [hematoxilin and eosin staining (H&E)]. A. Nulliparous control, B. Nulliparous PCOS, C. Uniparous control, and D. Uniparous PCOS. [hematoxilin and eosin staining (H&E)]. PCOS; Polycystic ovary syndrome.
Decrease in the number of secondary follicles (stars) in ovary of the rat model of polycystic ovary syndrome (PCOS) in comparison with the control rat [hematoxilin and eosin staining (H&E)]. A. Nulliparous control, B. Nulliparous PCOS, C. Uniparous control, and D. Uniparous PCOS. [hematoxilin and eosin staining (H&E)]. PCOS; Polycystic ovary syndrome.
Serum testosterone concentrations of nulliparous
the PCOS rats was more than the nulliparous control
(
The real-time PCR analysis showed that the expression
of
Hypertrophied and hyper-vaculated stromal cells in the ovarian medula of the polycystic ovary syndrome (PCOS) rats in comparison with normal stromal cells in the control rats [hematoxilin and eosin staining (H&E)]. A. Nulliparous control, B. Nulliparous PCOS, C. Uniparous control, and D. Uniparous PCOS. [hematoxilin and eosin staining (H&E)]. PCOS; Polycystic ovary syndrome.
Alterations of the mean (+SE) of serum hormone concentrations in
the female nulliparous and the primiparous rats after the exposition to
continuous light during 90 days for induction of polycystic ovary syndrome
(PCOS). A. Testestrone, B. Luteinizing hormone (LH), C. Follicle stimulating
hormone (FSH), and D. Decrease in the mean (+SE) expression of
a,b; Different letters show statistically significant differences between groups (P<0.05).
The present study for the first time showed that the
PCOS induction by constant light decreases
In women suffering from PCOS, the concentrations
of LH increases and FSH decreases in comparison with
healthy women (
Hyperandrogenism is accepted as an important attribute
of PCOS; therefore, in most animal models of
PCOS, androgen hormones have been used to stimulate
the PCOS (
Increase in serum concentrations of testosterone in
the PCOS rats of the nulliparous group compared to the
controls showed the efficiency of this model for evaluation
of PCOS in hypothalamus without exogenous androgens.
Consistent with our findings, increase in serum
testosterone levels of rats that were exposed to 112 days
constant light exposures with illumination intensity of
600 lux was shown (
To explain this phenomenon, it has been shown that
the testosterone concentrations are positively correlated
with the expression of the androgen receptor in the hypothalamic
suprachiasmatic nucleus (SCN). This locus
regulates circadian rhythms and light exposure controls
it (
Histopathologic evaluation of ovaries showed that
continuous light exposure increased the number of antral
follicles and atretic follicles. Consistent with our results,
increase in large antral and atretic follicles and reduction
of the number of early growing follicles have been previously
reported in rats that were subjected to 13 weeks
of continuous exposure (
However, the nulliparous group represented a better
PCOS model than the primiparous rats, but it is not clear
if gravidity can influence the occurrence of PCOS or
not. Consistent with the current finding, in the consistent
light model of PCOS, the PCOS criteria in the nulliparous
rats were more than the primiparous group. In
women, obesity would exacerbate the insulin resistance,
a predisposing factor for PCOS. It has been reported that
pregnancy can be a risk factor for obesity (
The constant light model of PCOS induced decrease in
the gene expression of