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JPCS Vol(6) ● Jan-March 2013 www.arpapress.com/Volumes/JPCS/Vol6/JPCS_6_02.pdf
8
INFLUENCE OF PROTEINS EXTRACT FROM HELIANTHUS ANNUUS
L. SEEDS ON BLOOD VOLUME OF REPRODUCTIVE ORGANS IN
PREGNANT MICE
Muhanad A. A. AlBayaty, Farid J.AL-Tahan & Huda F. Hasan
Dept. of Veterinary Physiological and Pharmacological, Collage of Veterinary Medicine, University of Baghdad.
ABSTRACT
L .arginine-NO pathway has been characterized as domination of blood volume and plays an important role in the
provocation of uterine function. Several sources of L.arginine, Helianthus annuus L. seeds had a great potential
dietary protein rich with L.arginine. The study protocol consist (420) pregnant mice separated into early and late
pregnancy groups each one were divided in to four groups: (1-crude extract of Helianthus annus, 2- partial purified
extracts of Helianthus annus, 3-L.arginine and 4-L.NAME) each 10 pregnant mice given the following dose
(100,150,200,250 and 300mg/kg B.W of crude extracts, purified extracts and L.arginine, L.NAME were given
(50,75,100,125 and 150) mg/kg B.W daily dose intraperitonally, finally normal salin were given to 20 pregnant mice
served as control each 10 for early and late respectively. The results displayed: 1- crude and partial purified protein
extraction of Helianthus annus contained L.arginine 35% and 85,4% respectively. 2- There were gradual
increase in blood volume of ovaries and uterus depend on dose increment and had highly correlated with increase of
hormones (estrogen and progesterone) of both extracts of Helianthus annus and L.arginine treated groups in early
and late pregnancy except in groups treated with dose 300mg/kg B.W. 3-The histology was illustrated the increase
blood vessels and vascular density of uterus and ovaries in each L.arginine and both Helianthus annus extracts
groups during late pregnancy. While in L.NAME groups had decrease in vascular density, blood vessels and micro
capillaries.
1. INTRODUCTION
Helianthus annuus plant is a strategic cultivated plant inIraq and the world (1). Several notions and reportes in
literatures revealed that Helianthus annuus are the seeds richest known source of the vital essential amino acid like
L.arginine (Madhusudhan,et al.,1999). L.arginine is considered as precursor of NO which had potential functions in
the uterus include vasodilatation and suppression of myometrial contractility during pregnancy (3).
The protocol of the present study was designed to shed the spots light on the effect of extracted L.arginine (crude
and purified ) from cheap source Helianthus annuus L , L.arginine and L.NAME on blood volume of pregnant mice
as possibility of maximizing of set points of fertility of breed which can be used as booster of local breeds fertility
characterization with preserving their biological function. This study was aimed to determine of whether differences
in utero-ovarian blood volume and uterine mass under influence of Helianthus annuus L.protein extract offering
various pharmacological profile develop uterine capacity and to examine the relationship between uterine blood
volume and l.arginine containing Helianthus annuus L.in early and late pregnancy.
2. MATERIALS AND METHODS Helianthus annuus L. Extraction and protein precipitation: 1-Preparation of the Helianthus annus seeds:( one
kilogram) were washed and dried then ground by electrical grinder. 2- Defatting of ground flower seeds powder by
petroleum ether in Sochxeltapparatus. 3- Crude protein extract according to method of (4). 4- Partial Purification
of protein to L.arginine according to method of (5 and 6). The concentration of L-arginine in partial purified protein
extract was determined according to (Bremer, et al.,2008 and Mc Donald, et al.,1997).
Experimental animals:
The total number of experiments reached to four hundred and twenty female mice, their weight was about (33-34 )g.
These mice grew up under same suitable condition at 21-24C◦, light (12) hrs. daily and kept in plastic cages were
cleaned daily, and food pellets and drinking water was presented adlibitum.
Protocol of experimental:eachgroups of (L.arginine ,crude extract and L.arginine extract of Helianthus annus )
was divided into five sub groups according to dose adminstration (100, 150, 200, 250and 300) mg/Kg B.W and
group of L.NAME was divided into five doses sub groups (50,75,100,125,150) mg/Kg B.W :
The first group: control group treated orally with normal saline.
The second group: crude extract of Helianthus annus treated orally for (1-6) days in early pregnancy and (7-19)
days in mid and late pregnancy.
JPCS Vol(6) ● Jan-March 2013 Al-Bayati et al. ● Reproductive Organs in Pregnant Mice
9
The third group: partial purified containing L.arginine extract of Helianthus annus treated orally for (1-6) days in
early pregnancy and (7-19) days in mid late pregnancy.
The forth group: L.arginine treated orally for (1-6) days in early pregnancy and (7-19) days in mid and late
pregnancy
The fifth group: L.NAME treated intrapretonially for (1-6) days in early pregnancy and (7-19) days in mid and late
pregnancy
EXPERIMENTAL PARAMETERS
Blood volume (in both early and late pregnancy experiments): The blood volume was determined by several
steps according to (7) blood sample was collected by a cardiac puncture at the time after scarified. Blood samples
(1ml) was diluted with 1ml of tris buffer (pH 7.4) and was homogenized and centrifuged. Supernatant was separated
and stored at -18C◦.The sample was thawed and diluted with 4ml Tris buffer (pH7.4). The absorbance was measured
by spectrophotometer, manually scanning of absorbance was done by increase wave length gradually and plotted the
absorbance wave length until sutible curve of blood absorbance was obtained.
Calculation of corrected blood Hb (∆A Two maximal peak (449) and (535) wave extracted from absorbance
curve, corrected absorbance (∆A) was calculated for tissue by the equsion as in figure (5) :
∆A= A449 – A535+A518
2
Figure (1): Absorbance curve of the heart blood, two maximal Peak at (449 and 535) nm, the elimination of the
irrelevant background of absorbance was also measured at 518 nm. This and subsequent figures of wavelength scans
were exact tracings of spectrophotometer printouts.
Step 2-Hb stain standard curve :
100mg of Hb stain was dissolved in 10 ml of distilled water with shaking then diluted to 0.5% before measuring the
absorbance. Calibration curve was done by using serial concentration of Hb. The optical density, the data was
resolved in regression between Hb and optical density, by using simple line regression
0
0.5
1
1.5
2
2.5
3
518
535
449
.
.
.
.
.
.
.
.
.
.
∆A
Hae
mo
glo
bin
Ab
sorb
tio
n (
AO
)
Wave length (nm)500
I550I
600I
650I
700I
JPCS Vol(6) ● Jan-March 2013 Al-Bayati et al. ● Reproductive Organs in Pregnant Mice
10
Figure (2): standard curve of Hb stain.
Step 3-Calibration curve of bloodvolume-Hb stain : 1ml of blood sample from heart was taken, this sample
was diluted with 1ml distilled water before measured the absorbance to give the first dilution, this procedure
repeated frequently until reaching to ten dilutions to give standard curve of heart blood, the diluted blood
samples were measured absorbance, these absorbance were calibrated in certain point in Hb-stain standard
curve (step 2) and extracted Hb concentration , then calibration curve Hb concentration (mg/ml) versus blood
volume was depicted.
Step 4-Ovarian and uterine Blood volume determination:
The ovary and uterus in early and late pregnant mice were excised from the body after two ligation from cervix and
ovarian terminal and blotted on a paper towel to remove blood from the surface, each of the uterus and ovary was
placed in (1ml) 0.05 M (hydroxymethyl) amino methane (tris) buffer (pH 7.4) and homogenized for 30 sec. in glass
homogenizer .The homogenate was centrifuged for 20 min. The supernatant fluids, which comprised each ovary or
uterus were transferred to polyethylene tube and stored at -18C◦ until a complete series of experimental samples
could be studied simultaneously. These samples were thawed and diluted 4-folds with Tris buffer (pH 7.4) before
their absorbance were measured by spectrophotometer. The absorbance value was setting in step 2 to check the
concentration of Hb and calibrated in step 3 to extracted blood volume.
Histological sectioning of ovarian and uterus tissue: Specimens of both ovaryand uterus (five specimens for dose 200mg/kg in each group) of animal were taken after
isolated from the other organs and kept in 10% formalin for fixation, processed routinely in histokinette, and
embedded in paraffin wax which cut at 5micrometer thickness by microtome and staind with haematoxylin and
eosin then examined under light microscope. (8).
Vascular parameters: They were measured according to (9):
1- Vascular density (VD): number of microvessels present in selected area, calculated as:
Micro-vessel number = Microvessel area+ residual stromal area X1000
2- Vascular area ratio (VAR): the area occupied by microvessels within the selected area.
Determined estrogen and progesterone levels: Determination the baseline of circulating serum levels of progesterone and estrogen (ten sample for each dose). The
hormonal measurements were done in each of treated and control pregnant mice groups. The quantitative analysis
was done in clinical Laboratory of Radio Active Isotope (Specific Al-Huda Laboratory).
Statistical analysis:
The ready program SAS (2001) was used in statistical analysis for study the control and treated groups was subject
to analysis two way of variance. A probability of P<0.05 was assumed to denote significant differences. LSD test
was used for comparison between groups and stages
Result Extraction: The sunflower seeds of Helianthus annus were extracted to yield approximately 10% of total solid and
the results crude extract contained 35% L.arginine and partial purified extract contained 85% of L.arginine,
Blood volume in uterus and ovaries: The blood volume showed in tables(1 and 2) significant (P<0.05) increase in
y = 0.280x + 0.054
R² = 0.986
0
0.5
1
1.5
2
0 1 2 3 4 5 6
abso
rban
ce (
AO)
Heamoglobin stian concentrations (mg/ml)
y=a+bx
JPCS Vol(6) ● Jan-March 2013 Al-Bayati et al. ● Reproductive Organs in Pregnant Mice
11
early and late pregnancy periods as compared with the control group, on the other hand, L.NAME groups displayed
a significant (P<0.05) decrease as compared with control groups, In other wise results displayed falling blood
volume rate in dose 300 mg\Kg during late period of pregnancy.
Table (1): The effect of L.arginine, crude extract , partial purified protein extract of Helianthus annuus seeds and
L.NAME in uterine Blood volume.(ml/gm) in pregnant mice with different doses during early and late periods of
gestation.
(1:A): In early pregnancy
(1:B): In late pregnancy
*Total number animals 10 / dose .
*Different capital letters mean significant difference (P<0.05)between column numbers.
*Different small letters mean significant difference (P<0.05)between row numbers.
*The control group in early pregnancy was same in all groups and also in late pregnancy
Groups
Doses
mg/kg
L.arginine treated
group
Helianthus annuus
crude extract
treated group
Helianthus annuus
partial purified
extract treated
group
Doses of
L.NAME
L.NAME treated
group
0 0.00238±0.0005 Aa 0.00238±0.0005Aa 0.00238±0.0005Aa 0 0.00238±0.0004Aa
100 0.00365±0.0003 Ba 0.00244±0.0004Ab 0.00311±0.0002Ba 50 0.000430±0.5495Bc
150 0.00539±0.0002 Ca 0.00341±0.0002Bb 0.00420±0.0001Cc 75 0.000147±0.0248Cd
200 0.01221±0.0002 Da 0.00726±0.0004Cb 0.01164±0.0001Da 100 -0.00198±0.0002 Dc
250 0.01208±0.0019 Da 0.01192±0.0003Db 0.01199±0.0001Dba 125 -0.00182±0.0001 Dc
300 0.01195±0.0001 Da 0.01047±0.0002Da 0.01138±0.0027 Da 150 -0.00176±0.0002 Db
Groups
Doses
mg/kg
L.arginine treated
group
Helianthus annus
crude extract
treated group
Helianthus annus
partial purified
extract treated
group
Doses of
L.NAME
L.NAME treated
group
0 0.02105±0.0004 Aa 0.02105±0.0004Aa 0.02105±0.0004Aa 0 0.02105±0.0004 Aa
100 0.02459±0.0003 Ba 0.02168±0.0002Bb 0.02292±0.0026Bc 50 -0.00530±0.0002Bd
150 0.03827±0.0002 Ca 0.02515±0.0001Cb 0.02720±0.0002Cc 75 -0.00196±0.0001 Cd
200 0.09091±0.0002 Da 0.00726±0.0004Cb 0.08903±0.0001Da 100 -0.00294±0.0002 Dc
250 0.09077±0.0001 Da 0.03403±0.0002Db 0.09070±0.0003Da 125 -0.00272±0.0002 Dc
300 0.08836±0.0003 Ea 0.09064±0.0005Eb 0.08796±0.0002Aa 150 -0.00286±0.0004 Dc
JPCS Vol(6) ● Jan-March 2013 Al-Bayati et al. ● Reproductive Organs in Pregnant Mice
12
Table (2) The effect of L.arginine , crude extract , partial purified protein extract of Helianthus annus seeds and
L.NAME in ovaries Blood volume(ml/g) in pregnant mice with different doses during early and late periods of
gestation.
(2:A): In early pregnancy
(2:B): In late pregnancy
*Different small letters mean significant difference (P<0.05)between row numbers.
*The control group in early pregnancy was same in all groups and also in late pregnancy.
Groups
Doses
mg/kg
L.arginine treated
group
Helianthus annus
crude extract
treated group
Helianthus annus
partial purified
extract treated group
Doses of
L.NAME
L.NAME treated group
0 0.00032±0.0002 Aa 0.00032±0.0002Aa 0.00032±0.0002 Aa 0 0.00032±0.0002 Aa
100 0.00124±0.0012 Ba 0.00057±0.0002Bb 0.00097±0.0001 Bc 50 -0.00530±0.0002 Bd
150 0.00499±0.0016 Ca 0.00169±0.0001Cb 0.00432±0.0011 Cc 75 -0.00196±0.0013 Cd
200 0.00914±0.0011 Da 0.00580±0.0016Db 0.00898±0.0014 Da 100 -0.00294±0.0017 Dc
250 0.00893±0.0017 Da 0.00818±0.0021Ea 0.00861±0.0014 Da 125 -0.00242±0.0011 Db
300 0.00903±0.0001 Da 0.00803±0.0001Ea 0.00836±0.0018 Da 150 -0.00216±0.0017 Db
Groups
Doses
mg/kg
L.arginine treated
group
Helianthus annus
crude extract
treated group
Helianthus annus
partial purified
extract treated
group
Doses of
L.NAME
L.NAME treated
group
0 0.00218±0.0005 Aa 0.00218±0.0005Aa 0.00218±0.0005Aa 0 0.00218±0.0005 Aa
100 0.00263±0.0011 Ba 0.00250±0.0002Bb 0.0025±0.0002 Bc 50 -0.00345±0.0001Bd
150 0.00478±0.0014 Ca 0.00301±0.0016Cb 0.00405±0.0002Cc 75 -0.00388±0.0001 Cd
200 0.05689±0.0022 Da 0.01100±0.0017Db 0.05598±0.0026Da 100 -0.00462±0.0011 Dc
250 0.05650±0.0011 Da 0.05570±0.0013Eb 0.05584±0.0045Da 125 -0.00430±0.0001 Dc
300 0.05300±0.0071 Ea 0.09064±0.0005Eb 0.08796±0.0002Aa 150 -0.00286±0.0004 Dc
JPCS Vol(6) ● Jan-March 2013 Al-Bayati et al. ● Reproductive Organs in Pregnant Mice
13
progesterone and estrogen hormones during early and late period of pregnancy mice :The result in (tables 3
and 4) showed that levels of progesterone and estrogen increased significantly (P<0.05) in treated groups (L.arginine
and both extracts) during early and late period of pregnancy as compared with the control groups in addition the
estrogen controversially was gradual decrease in as flow up of increase dose, while L.NAME groups showed a
significant (P<0.05) decrease of estrogen and progesterone hormones as compared with the control, L.arginine and
both extracted groups.These results showed a significant (P< 0.05) increase in estrogen hormone in dose 300mg/kg
of Larginine and purified extract during late period of pregnanc.y
Table (3): The effect of L.arginine , partial purified extract and L.NAME on progesterone hormone (Pg/ml) with
different doses in early and late period of pregnancy:
(3:A): In early pregnancy
(3:B): In late pregnancy
* Different capital letters mean significant difference (P<0.05)between column numbers.
*Different small letters mean significant difference (P<0.05)between row numbers.
*The control group in early pregnancy was same in all groups and also in late pregnancy.
Groups
Doses
mg/kg
L.arginine treated
group
Helianthus annus partial purified
extract treated group
Doses of
L.NAME
L.NAME treated group
0 1.93±0.08Aa 1.93±0.08Aa 0 1.93±0.08Aa
100 2.00±0.34Ba 1.95±0.29Ab 50 0.71±0.15Bc
150 2.37±0.44Ca 2.10±0.51Ba 75 0.26±0.61Bb
200 2.68±0.63Da 2.30±0.31Ba 100 0.09±0.36Cb
250 2.70±0.60Da 2.40±0.44Ba 125 0.03±0.62Cb
300 2.85±0.37Da 2.5±0.52Ba 150 0.01±0.04Cb
Groups
Doses
mg/kg
L.arginine treated
group
Helianthus annus
partial purified
extract treated
group
Doses of
L.NAME
L.NAME treated
group
0 2.93±0.17Aa 2.93±0.17Aa 0 2.93±0.17Aa
100 2.98±0.08Aa 2.95±0.07Aa 50 0.45±0.31Bb
150 3.65±0.15Ba 3.50±0.27Ba 75 0.10±0.45Bb
200 5.94±0.37Ca 4.8±0.11Ca 100 0.06±0.50Cb
250 5.97±1.81Ca 4.9±0.98Ca 125 0.03±0.91Cb
300 1.8±0.33Da 1.7±0.61Da 150 0.02±0.09Cb
JPCS Vol(6) ● Jan-March 2013 Al-Bayati et al. ● Reproductive Organs in Pregnant Mice
14
Table (4):The effect of L.arginine , partial purified extract and L.NAME on estrogen hormone (Pg/ml) with different
doses in early and late period of pregnancy:
(4:A): In early pregnancy
(4:B): In late pregnancy
Angiogensis and vascular density:The results appeared an increase of angiogenesis, and vascular density in uterus
and ovaries listed in (tables 5 and 6) respectively.
Table (5): The effect crude and partial purified protein of Helianthus annus, L.arginine and L.NAME on blood
vessels (angiogenesis) during late pregnancy:
Groups
Doses
mg/kg
L.arginine treated
group
Helianthus annus
partial purified
extract treated
group
Doses of
L.NAME
L.NAME treated
group
0 4.63±0.38Aa 4.63±0.38Aa 0 4.63±0.38Aa
100 8.96±1.30Ba 8.50±1.62Ba 50 1.7131±2.70Bb
150 8.25±1.16Ba 8.00±0.99Ba 75 0.63±1.30Cb
200 6.07±0.84Ca 6.30±0.74Ca 100 0.23±0.98Db
250 5.90±5.90Ca 6.20±6.09Ca 125 0.08±0.53Db
300 5.26±0.20Ca 5.50±0.64Ca 150 0.03±0.71Db
Groups
Doses
mg/kg
L.arginine treated
group
Helianthus annus
partial purified
extract treated
group
Doses of
L.NAME
L.NAME treated
group
0 2.06±0.21Aa 2.06±0.21Aa 0 2.06±0.21Aa
100 5.38±1.00Ba 4.9±2.16Ba 50 0.49±1.73Bb
150 5.01±0.92Ba 4.5±0.88Ba 75 0.11±2.18Cb
200 4.71±0.67Ca 4.1±0.85Ba 100 0.04±0.72Db
250 4.0±1.08Ca 4.0±0.99Ba 125 0.02±0.74Db
300 9.5±0.31Da 9.3±0.61Ca 150 0.01±0.90Db
Control treated group L.NAME
treated
groups
L.arginine
treated group
Partial
purified
extract treated
group
Crude extract
treated group
Blood vessels
7.16 X106
±
5.81Ad
3.87 X 106
±
0.10Ac
26.61 X106
±
5.21Ab
27.04 X106
±
1.80Ab
23.2 X106
±
2.59Aa
Uterine area
6.26 X 106
±
d 1.88B
2.64 X 106
±
0.20Bc
18.80 X 106
±
0.93Bb
16.51 X 106
±
0.65Bb
14.8X106
±
2.07Ba
Ovarian area
JPCS Vol(6) ● Jan-March 2013 Al-Bayati et al. ● Reproductive Organs in Pregnant Mice
15
Table (6): The effect crude and partial purified protein of Helianthus annus, L.arginine and L.NAME on vascular
density during late pregnancy
Figures (3): The uterine Cross section (B) of L.arginine treated groups, (C) of partial purified extract of Helianthus
annus treated group and (D) , crude extract of Helianthus annus treated groups display distribution of blood vessel
and micro blood vessel in endometrium and myometrium and branches with high density of vascularization, and
showed different of micro capillary of arterioles of endothelium cells. Showed flatten cell contain RBCs with
association invasive pattern of endothelial cell to new formation arterioles. In (A) control treated group showed
normal distribution of blood vessel and less vascularization density while in (E) L.NAME treated groups showed
decrease blood vessel in endometrium and very low of vascular density . (Heamatoxiline and Eosin).
A X225
B X225
Control treated
group
L.NAME
treated
groups
L.arginine
treated group
Partial purified
extract treated
group
Crude extract
treated group
Vascular
density
7.59±1.36d
3.67±0.12c 23.60±2.58b 21.81±1.89b
14.58±1.62a
Uterine
area
JPCS Vol(6) ● Jan-March 2013 Al-Bayati et al. ● Reproductive Organs in Pregnant Mice
16
C X225
D X225
E X 225
JPCS Vol(6) ● Jan-March 2013 Al-Bayati et al. ● Reproductive Organs in Pregnant Mice
17
Figures (4): A cross section in ovary A2) represented L.ar ginine treated group , (B2) partial purified extract of Helianthus
annus treated group and (C2) crude extract of Helianthus annus treated group display distribution of blood vessel and micro
blood vessel and branches with high density of vascularization , and showed different of micro capillary of arterioles showed
flatten cell contain RBCs with association invasive pattern of endothelial cell to new formation arterioles and (D2)
represented control treated group indicated normal distribution of blood vessel while (E2) L.NAME treated group showed
decrease in blood vessel and vascularization density to induced sinusoid (Heamatoxiline and Eosin).
A2 X225
B2X 225
C2X 225
JPCS Vol(6) ● Jan-March 2013 Al-Bayati et al. ● Reproductive Organs in Pregnant Mice
18
D2X 225
E2X 225
DISCUSSION The extraction: the results of crude extract and partial purified extract agreed with those reported by Pearce, (4).
The blood volume:The results of increase blood volume of uterus and ovaries in both proteins extract and
L.arginine treated groups this might be attributed to three important mechanism of L.arginine-NO pathway: 1-
stimulation of β-adrenergic receptors in the uterine vascular beds which reduced basal and agonist-stimulation
inositol phosphate production through activation of K+ channels, associated with increasing cGMP and beside that
the production of cGMP may be due to increase in nitric oxide as a metabolite of L.arginine, all these led to produce
an increased in uterine blood volume this result agreement with (10).
2- regulate cytosolic [Ca++
] lead to the vasodilation increase blood volume was demonstrated by (11).
3-increase progesterone hormone might regulate the cGMP effector system for relaxation in both the myometrium
and blood vessels led to increase blood demined through enlarging the blood vessel capacity then blood volume
(12) .
The gradual increase in blood volume of uterus and ovaries of crude extract indicated that Helianthus annus seeds
might be due to nutritional quality (13) and their potential as dietary protein sources in animal feed is well
recognized (14).Helianthus annus seeds also contained lecithin, tocopherols, carotenoids, waxes and high vitamin E
content (15).
The falling blood volume rate in dose 300 mg\Kg during late period of pregnancy, these results might be explained
by accumulation of free radical (nitrite) in blood by an extremely increase in oxidation of NO to N2O3 due to over
dose causing tissue injury, toxic effect and abortion, these explained agreement with (16). other studies showed a
proportional relationship between abortion and rise of estrogen level and decrease progesterone which agreement
with our result in dose 300mg/kg of Larginine and purified extract during late period of pregnancy(17).
The decrease in blood volume of dose 300mg/kg of crude extract during late periods of pregnancy might be
attributed to the presence of other compounds as like trypsin inhibitor (anti-nutritive factor) had been observed in
Helianthus annus seedsthat presumably might be effect although the ratio of this inhibitor was low, (18).
The decrease in blood volume of uterine and ovaries during early and late pregnancy of NG-nitro-L-arginine methyl
ester L.NAME groups this might be due to direct effect of (L-NAME) on NOS inhibition which prevent conversion
of L-arginine to citrullin which neglected NO relaxing effect lead to which provoke PGF2α that led to stimulate Ca++
JPCS Vol(6) ● Jan-March 2013 Al-Bayati et al. ● Reproductive Organs in Pregnant Mice
19
entry into smooth muscle cells through L-type Voltage-operated Ca channel (VOCCs) and increase in the sensitivity
of contractile apparatus to Ca (19)
The progesterone and estrogen hormone: The high progesterone hormonal level in both protein extracts and
L.arginine treated groups might be attributed to NO decrease prostaglandin F2α (PGF2α)-induced inhibition in
progesterone in ovaries by block the receptors of PGF2α in granulosal cell which appear to mediate the luteolytic
actions of this hormone, attenuating the fall in the serum progesterone concentration (20).
During early pregnancy estrogen might elevate eNOS expression in the myometrial tissues via estrogen receptor
ERα and/or ERβ, and that this elevation of eNOS may play some roles in the maintenance of the pregnancy, this
might explained the increase which happened in estrogen (21).
The gradual decrease in estrogen in treated groups might be attributed to that NO donors inhibited both estradiol
secretion and aromatase activity. Further, aromatase activity in microsomal of granulosa cells was inhibited by
native NO. (22)
The changes of L-NAME groups result caused a decrease in progesterone and estrogen level might be due to
impaired NO production which provoke PGF2α stimulant in order to PGF stimulation led to stimulate Ca++
entry into
smooth muscle cells through L-type Voltage-operated Ca channel (VOCCs) and decrease in progesterone hormone
(19).
Histophysiology: The increase angiogenesis parameter might be attributed to mechanisms associated with
L.arginine-NO pathway was enhanced endothelial cell proliferation, perhaps by increasing the expression of
vascular endothelial growth factors, this pathway also enhances endothelial migration by stimulating endothelial
cells podokinesis, enhancing the expression of α and β receptors and increasing dissolution of the extracellular
matrix via the basic fibroblast growth factor–induced upregulation of urokinase-type plasminogen activator (23).
The increased vascularity of dominant follicles which stimulated by L.arginine-NO pathway attributed to increase
serum gonadotropins (24) in addition to positive relation of uterine and ovarian blood volume (25).
The lowest angiogenesis and vascular density in Helianthus annus crude extract this might attributed to presence of
anti-nutritive factor and ratio of unsaturated fat like trypsin that decrease the blood volume and decrease corpus
luteum angiogenesis.
The decrease of uterine and ovaries angiogenesis in L.NAME groups might be attributed to inhibition of the NOS
which had a stimulatory role of NO in angiogenesis and blocking of vasoactive molecules such as substance P and
prostaglandin E1 (Ziche, et al., 1994).
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