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ResearchArticle
EFFECTOFATROPINESULPHATEONOVARIANACTIVITIESINALBINORATS
MADHUM.PATIL1,SHARANGOUDAJ.PATIL*2ANDSARASWATIB.PATIL1
ReproductiveBiologylaboratory,DepartmentofZoology,GulbargaUniversityGulbarga585106,Karnataka,India,2Toxicology
Laboratory,BioenergeticsandEnvironmentalScienceDivision,NationalInstituteofAnimalNutrition&Physiology(NIANP)
Adugodi,Bangalore560030,Karnataka,India.Email:shajapatil@gmail.com
Received:22Dec2009,RevisedandAccepted:27Jan2010
ABSTRACT
Atropinesulphateatthedoselevelof0.1mg&0.2mg/100gmbodyweightadministrationfor30daystothecyclingalbinorats,causeddecreasein
theovarianweight,showingadecreasingnumberofdevelopingfollicles,Graafianfolliclesandcorporalutea,andanincreasednumberofatretic
follicles in histological sections. The estrous cycles of these rats were irregular with prolonged diestrus and reduced proestrus, estrus and
metaestrusphasesalsosupportthedecreasedestrogensynthesis.ResponsibleforcornificationofvaginalsmearinAtropinesulphatetreatedrats.
The hisometric changes of diameter of the ovarian follicles are reduced significantly. The total cholesterol content of the ovary was increased;
proteinandglycogencontentweredecreased.
Keywords:Atropinesulphate,Rats,Ovary,Graafianfollicle,Atreticfollicle,Corporalutea,Estrouscycle.
INTRODUCTION
Atropine, is a naturally occurring alkaloid of plant Atropia
belladonna. The other sources are Dattura inoxia, Dattura
stramonium.Itisacompetitiveantagonistofmuscarimiccholinergic
drug. Generally, Atropine sulphate is used as atropine sulphate
injection and chemically designed as 1H, 5HTropan3OL ()
tropate (ester) sulphate (2:1) (salt) monohydrate, (C17H23NO3)2
H2SO4H2O1.Asinglesubcutaneousinjectionofatropineonproestrus
day delays ovulation for several hours in mice2. The studies of
Redmond3 indicate that, the atropine effectively blocks the
progesterone induced ovulation in rats. In male rats the
administration of this drug into autonomic nerve inhibits the
testicular development4. All the facets of activity exhibited by
nervous system are susceptible to pharmacological manipulation.
The anaesthetic gases, the aliphatic alcohols, the barbiturates, the
nicotine and atropine, interfere in the activities of the CNS therapy
modify the action of the gonads and associated organs. CNS
depressants acts on the hypothalamus and inhibit the release of
gonadotrophin releasing hormone (GnRH) and corticotrophin
releasingfactor(CRF)thusdecreasingthecirculatingconcentrations
of luteinizing hormone (LH), follicle stimulating hormone (FSH),
adrenocorticotropichormone(ACTH)andendorphin5.Secretions
of pituitary gonadotrophins are regulated by brain and neurons
situated in the anterior parts of the hypothalamus that synthesize
theGnRH6.AccordingtoseveralinvestigatorsCNSinfluencingdrugs
inhibitthereleaseofFSHandLH fromthepituitaryactionthrough
hypothalamus, blocking the neural stimulus to the gonadotrophin
releasing hormone79. Though there are many indirect evidences of
atropinesulphateonreproduction,sofar,nodirectactionhasbeen
reported.Therefore,inthepresentstudyisaimedtounderstandthe
effect of Atropine sulphate on ovarian activities which are
dependentonhypophysicalgonadotrophinsinalbinorats10,11.
and 12h darkness: humidity: 5055%). The rats were fed with
balanced diet as per CFTRI, Mysore, INDIA formula and water ad
libitum.Theratsweredividedintothreegroupsofsixanimalseach.
Group1:Received0.2mlsaline/100gbodyweightfor30days.
Group 2: Received 0.1mg Atropine sulphate in 0.2ml saline/100gm
bodyweightfor30days.
Group 3: Received 0.2mg Atropine sulphate in 0.2ml saline/100gm
bodyweightfor30days.
The treatment was started from estrus phase only, as the ovarian
activities changes markedly from one phase to another phase of
oestrous cycle. The saline or Atropine sulphate was administrated
intraperitoneallyeverydaybetween10:00to11:00AM
All the rats were sacrificed on 31st day, 24 hour after the last
treatment. The ovaries were dissected out immediately and
separatedoutfromtheadherenttissueandweighedtothenearest
mg on an electronic balance. Organ from one side of each rat were
fixed in Bouins fluid, embedded in paraffin wax, sectioned at 5m,
stained with haemotoxylineosin for histological studies. Ovarian
follicular diameter and morphologies were used to classify follicles
byusingestablishedmethod12,13.Morphometricstudiesoftheovary
were made by using stage and ocular micrometer and organ from
the other side was used for biochemical estimations like protein14,
glycogen15andcholesterol16.
RESULTS
Changesinthebodyweight[Table1]
There is nonsignificant change in the body weight after
administrationofAtropinesulphate.
MATERIALSANDMETHODS
ChangesintheOvary
Animals
Gravimetricchanges[Table2]
Table1:EffectofofAtropineSulphateonthebodyweightofalbinorats
Weightof
Treatment
Initialbodyweight Finalbodyweight
%Increase
theOvary
168.960.97
Saline
155.252.95
8.83
39.571.91
Atropinesulphate
157.421.80
170.372.85
8.22
33.411.01*
(0.1mg/100gbodywt.)
Atropinesulphate
(0.2mg/100gbodywt.)
153.762.54
165.232.70
7.46
21.441.10**
Duration:30days,6animalsaremaintainedineachgroup.MS.E.=MeanStd.Error,*=P<0.05,**=P<0.01,***=P<0.001
Biochemicalchanges[Table2]
TheAtropinesulphateadministrationhasshowninhibitoryeffecton
ovarianactivities.Cholesteroltheprecursorforsteroidbiosynthesis
isincreasedsignificantly(p<0.01)with0.1mgandhighlysignificant
(p<0.001) with 0.2mg of Atropine sulphate administration. The
%Inhibition
__
15.56
45.81
Table2:EffectofAtropineSulphateontheBiochemicalchangesofOvary
Weightof
Cholesterol
Protein
Glycogen
Treatment
ovary
(g/mgovary)
(g/mgovary)
(g/mgovary)
Saline
39.571.91
27.501.37
23.530.43
6.130.29
Atropinesulphate
33.411.01*
32.280.63**
18.090.38**
3.050.13***
(0.1mg/100g body
wt.)
Atropinesulphate
21.441.10**
36.210.30***
15.750.35***
2.080.16***
(0.2mg/100g body
wt.)
Duration:30days,6animalsaremaintainedineachgroup.MS.E.=MeanStd.Erro,*=P<0.05,**=P<0.01,***=P<0.001
nonsignificantly with 0.1mg and significantly (p<0.05) and with
0.2mg doses, the highly significant reduction (p<0.001) with both
the doses and number of corpora lutea which are formed after the
ovulation were decreased significantly (p<0.01) with 0.1mg and
highly significantly (p<0.001) with 0.2mg of Atropine sulphate
administration. The regressing follicles like atretic follicles were
increasedalmostsignificantly(p<0.05)with0.1mgandsignificantly
with0.2mgofAtropinesulphateadministration.
Histologicalchanges[Table3;Figure13]
The histological sections of the ovaries of Atropine sulphate
administration has decreased in number of healthy follicles and
increasedinthenumberofregressingfollicles.
The number of healthy follicles like primary follicles has reduced
significantly(p<0.01)with0.1mgandhighlysignificantly(p<0.001)
with0.2mgdoses.Thedecreaseinthenumberofsecondaryfollicles
Table3:EffectofAtropineSulphateontheHistologicalchangesofOvary
Treatment
PrimaryFollicles
SecondaryFollicle
Saline
3.800.24
3.500.16
GraafianFollicles
AtreticFollicles
3.700.15
2.200.24***
1.300.15
1.670.16*
Atropinesulphate
2.900.23*
3.400.21
(0.1mg/100g body
wt.)
Atropinesulphate
Duration:30days,6animalsaremaintainedineachgroup.MS.E.=MeanStd.Error,*=P<0.05,**=P<0.01,***=P<0.001
Corpora
Lutea
4.700.82
3.500.16**
3.100.23***
94
Fig. 1: Photomicrograph of ovary treated with
vehicle showing normal fully developed primary,
secondary follicles and Graafian follicle with
healthyoocyte(x100).
Histometricchangesofovariancomponents[Table4;Figure13]
Changesintheoestrouscycle[Table5]
Treatment
PrimaryFollicles
SecondaryFollicle
GraafianFollicles
AtreticFollicles
Saline
9.090.09
26.470.28
36.160.88
34.120.25
39.200.21
7.190.14*
21.940.37*
29.020.18*
31.020.24*
34.020.24*
5.970.27**
15.030.30**
21.080.21**
28.140.29**
29.200.21**
Atropinesulphate
(0.1mg/100gbodywt.)
Atropinesulphate
(0.2mg/100gbodywt.)
Corpora
Lutea
Duration:30days,6animalsaremaintainedineachgroup.MS.E.=MeanStd.Error,*=P<0.05,**=P<0.01,***=P<0.001
95
Table5:EffectofAtropineSulphateonthedurationofvariousstagesofEstrousCycleinalbinorats
Treatment
Proestrus
Estrus
Metaestrus
Diestrus
Saline
4.620.30
4.210.31
3.980.06
18.080.32
Atropinesulphate
2.890.18**
2.561.42***
1.980.14***
21.971.28***
(0.1mg/100gbodywt.)
Atropinesulphate
2.340.24***
2.090.12***
1.870.02***
24.011.68***
(0.2mg/100gbodywt.)
Duration:30days,6animalsaremaintainedineachgroup.MS.E.=MeanStd.Error,*=P<0.05,**=P<0.01,***=P<0.001
DISCUSSION
In the present investigation the weight of ovary is reduced
significantly due to the administration of Atropine sulphate. As the
drug is administrated between 10 to 11:00 AM every day, there is
possibility of covering the so called Critical period for cyclic LH
surge,necessaryforovulation;thuspostponingtheovulationforone
day by interfering with 24 hours periodicity for gonadotrophin
release17,18. Low levels of plasma FSH and LH with high
concentrationofpituitarygonadotrophinandprolactinareobserved
afterAtropinesulphateadministrationbysomeinvestigators7,9.Itis
well known that hypothalamus regulates the rhythmic release of
pituitary gonadotrophin i.e., FSH, LH and prolactin through the
neuralstimulustogonadotrophinreleasinghormone(GnRH)19.The
orderly event of follicular growth and ovulation depends upon the
pituitary FSH, LH & prolactin. FSH stimulates the differentiation of
granulosacellsandpromotesthefolliculardevelopment 2022.
In the present study the ovaries of treated rats have reduced
significantly with retarded follicular growth, differentiation of
granulosa cells in the follicles, underdeveloped follicles and
reduction in the ovulatory follicles may be attributed to the non
availability of gonadotrophin and these are very essential for
maintenanceofovarianactivities.
The continuous presence of FSH within the follicles prevents the
follicle undergoing atresia23. The large number of atretic follicles
along with degeneration of granulosa cells and disappearance of
antruminAtropinesulphatetreatedratsmaybeduetoinadequate
supplyofpituitaryFSH.Asovulationneedsincreasedconcentration
ofplasmaLHandFSH2426.Atropinesulphatemighthaveresultedin
the inhibition gonadotrophin release resulting in the blockade of
ovulation as evidenced by decreased in the number of freshly
formedcorporalutea.
9.
10.
12.
11.
13.
14.
15.
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