Document Type : Original Article
1 Department of Biology, Faculty of Basic Sciences, Shahed University, Tehran, Iran
2 Department of Pathology, School of Medicine, Shahed University, Tehran, Iran
3 Department of Embryology, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran
Polycystic ovarian syndrome (PCOS) is introduced
as common endocrine disorder that affects up to 10%
of women during their reproductive ages (
NO is indicated as an important paracrine messenger
that participates in several physiological and
pathophysiological events in the endocrine organs (
This study evaluates the influence of L-arginine, a precursor of NO, in PCOS-induction in female Wistar rats. NADPH-diaphorase provides valuable biochemical data representing the participation of NO in this phenomenon. The marker is classified as the best indicator of NOS enzyme. We have also examined the metabolic status in L-arginine-exposed rats and evaluated the effect of naloxone in L-argininetreated rats.
In this experimental study, the animals were
eight-week-old female Wistar rats (weight:
200-250 g) purchased from Pasteur Institute
of Iran (Tehran, Iran). Animals were retained
under standard conditions in accordance to the
Guide for the Care and Use of Laboratory Animals
L-arginine, nitroblue tetrazolium (NBTS), and β-nicotinamide adenine dinucleotide phosphate (β-NADPH) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Naloxone hydrochloride was provided by the Tolid Daru Co., Tehran, Iran. Ketamine and xylazine were obtained from Veterinary Organization, Tehran, Iran.
Female rats have a 4-5 day cycle, including
proestrous (12 hours follicular growth and peak estrogen
phase), estrous (12 hours ovulation phase),
metestrous (12 hours corpus luteum secrets progesterone
phase), and diestrous (48 hours corpus
luteum regression phase). Without exposing with
male rats, the female rats are always in the diestrous
phase; their phases will progress only
after mating with a male rat (
Female Wistar rats (200-250 g) at eight weeks of age were randomly divided into the L-arginine dose groups (50-200 mg/kg; n=10 in each) and a group that was pre-injected with naloxone (0.4 mg/kg; n=10) prior to L-arginine administration. The drug, L-arginine, was injected intra-peritoneally (i.p.) once daily during a period that ranged from 9 to 14 days. Naloxone was injected once per day 30 minutes prior to L-arginine administration. The control group received saline (1 ml/kg i.p.) instead of the drugs.
The treatment groups were anesthetized by diethyl ether in a special device. Then a 2 cm midline incision in the lower abdomen area was performed. The ovaries were carefully examined biometrically and dissected out, then collected in a fixative for further histological and biochemical analysis.
For histological investigations, the ovaries were
fixed in a 10% formalin solution and processed
with a tissue processor through paraffin embedding.
Serial sections (3-5 μm) were prepared with
a rotary microtome. The slides were then stained
using the hematoxylin and eosin (H&E) staining
At the end of the experiment, the rats were anesthetized
by i.p. injections of 100 mg/kg ketamine
hydrochloride and 20 mg/kg xylazine. Heart blood
samples were obtained between 8:00-10:00 am after
an overnight fast to assess sugar, lipid, and hormonal
profiles. All samples were kept at room temperature
for at least 30 minutes to facilitate the clotting of the
samples. The samples were then centrifuged at 3000-
5000 g for 15 minutes. The sera were stored at −20˚C
for future assessments. The level of sera agents
[high-density lipoprotein (HDL), low-density lipoprotein
(LDL), glucose, cholesterol, and triglycerides]
were measured based on competition binding
using the enzyme-linked immunosorbent assay (ELISA)
and read with an ELISA reader. Other tests were
performed by using a Cobas Mira plus CC Chemistry
Analyzer as previously mentioned (
The activation of nitric oxide synthase (NOS)
enzyme in the ovarian tissue was shown using the
NADPH-diaphorase at the histochemical level.
Rats were anesthetized 24 hours after the last injection,
and then the ovaries were excised and trimmed
from periovarian fat and bursae. The samples were
then immersed in phosphate buffered saline (PBS,
pH=7.2) overnight at 4˚C, followed by cryoprotection
in 30% sucrose. Then specimens were sectioned
with cryostat microtome at -15˚C (9-10 μm frozen
sections) and mounted on poly-L-lysine-coated slides
The tissue slides were observed under the videophotolightmicrocope (Olympus, USA and the provided images were then assessed in 100-μm2 units of the photorecords at ×4 or more magnification using the Image Tool program (UTHSCSA, version 2.03), the free image processing and analysis program for Microsoft Windows to provide quantification for an area of 100 μm2.
Data were analyzed by the Kolmogorov–Smirnov (K-S) test for showing the equality to analysis by variance (ANOVA) using SPSS software (version 13.0; SPSS Inc., Chicago, IL). A Tukey-Kramer or LSD post hoc test was used to calculate the difference between the groups after ANOVA. Statistical significance was considered p<0.05. All data were expressed as mean ± SEM. In case of the NADPHdiaphorase technique, the intensity analysis was assessed at 100 μm2 using the Image Tool program (UTHSCSA, version 2.03), after calibrating for a 100 μm2 area.
The estrous cycle phase of female virgin rats was
determined as diestrous after using the PAP stain
Result of Papanicolaou (PAP) stain of female virgin rat vaginal smears in the diestrous phase. The abundance of leukocytes in the smear samples indicate that the cycle phase is diestrous.
The rats’ ovaries from the L-arginine dose
groups (50-200 mg/kg) exhibited small follicles
in the early development phase in addition
to the follicles that showed evidence of either
atresia, large cysts with thickened granulosa
cell layer, or large cystic follicles with scant
granulosa cells (
Pictures of ovaries from control (a, A), solely L-argininetreated (B), and naloxone pre-treated (C) rats. Bars beside the samples show mm values. The figures are shown under magnification [a: ×1.6 objective of an (inverted) photomicroscope; A, B, C: Olympus photomicroscope at × 4].
Positive NADPH-diaphorase reactivity was observed
in the stroma and in the theca cell layer in the
ovaries of L-arginine treated rats (
Histochemical NADPH-diaphorase evaluated in control (A) and treated rats; single L-arginine (B) or L-arginine + naloxone (C) as detailed in materials and methods.
Levels of serum parameters are shown in
Curves indicate the levels of metabolic agents in serum (mg/dL) both in the control and experimental groups (n=10). The experimental rats received L-arginine (50 mg/ kg, i.p., 14 days) or L-arginine (50 mg/kg, i.p., 14 days) plus naloxone (0.4 mg/kg, i.p., 14 days). Naloxone was injected 30 minutes prior to L-arginine. Values are mean ± SEM. * p<0.05 vs. control (Tukey or LSD test).
NO is categorized as an important intra-ovarian mediator
This study provided more evidence for follicular
atresia, as well as the production of large cysts due to
NO treatment in Wistar rats, which agreed with a previous
finding. Despite the vascular effect of NO, which
forms the highly reactive cytotoxic peroxynitrite (
Except for the level of serum LDL (
Though no significant change in sera levels was found,
the administration of naloxone prior to L-arginine led to
a decrease in the level of serum LDL (
The findings show a correlation between central opioid tone and body weight. The regulation of metabolic agents by naloxone in this study beyond the effect of the drug on PCOS signs remains elusive.