The Effect of Repaglinie and Metformine on Ovarian Function of Alloxan Induced Diabetes Female Virgin Rats
ShaymaaAbdAl-jasimAlshukri1a,FarisNajiAboodAlhady2,AmelAliAltee2 1BiotechnologyDepartment,CollegeofBiotechnology,Al-
QasimGreenUniversity.2BiologyDepartment,CollegeofScience,UniversityofBabyl on
a]Correspondingauthor:[email protected] ABSTRACT.
There is association between diabetes mellitus and abnormal ovarian function. There are many oral hypoglycemic agents that are used to treat diabetes as repalinide and metformin . To estimate the effeciancy of repalinide and metformin in improvement ovarian function virgin female ratswere used in this study.The female divided into two groups the first group were used for the induction of diabetes mellitus typeІІ using alloxan monohydrate. The rats with continual blood glucose level above 200 mg/dl for seven day were considered to be hyperglycemic and were selected for treatment,the second group of female rat were given normal saline intraperitonialy, both two female groups were divided into three subgroups that included. Normal control, Non-diabetic rats treated with repaglinide 4mg/kg/, Non-diabetic rats treated with metformin 1000 mg/kg/day, Diabetic control (DC) ,Diabetic rats with repaglinid 4mg/kg/day and Diabetic rats with metformin 1000 mg/kg/day. After 6 weeks of treatment rats were sacrificed , ovaries were collected and then weighted.Subsequently, histological sections of overies were prepared to investigate the mean total number of follicles and corpus lutea in control and treatment groups to evaluate the effect of treatment on ovarian function. The results revealed significant increase (P≤0.05) of ovarian weight in diabetes with repaglinide group and significant increase (p≤0.05) in secondary follicles number and corpus luteum number in normal with metformin group as compared to normal control group
Keywords:Diabetes mellitus, ovarian function , repalinide ,metformin, number of follicles.
INTRODUCTION
Diabetesmellitusinducedabnormalovarianfunctionviatriggeringapoptosisofgranulosace llsandsuppressedovarianangiogenesisleadingtoabnormalfolliculardevelopmentandcorp usluteumformation.thereforeeffectiveglucosecontrolisnecessaryfornormalovarianfunct ionindiabeticmellitus[1].Diabetesmellitustype2greatlyaffectsgonadotrophinshormones (FSHandLH)inbothmenandwomen[2].Gamete(spermoregg)productionstimulatesbyFS H,whileproductionofgonadalhormonespromotesbyLH[3].Repaglinideisainsulinsecreta goguethatlowerspostprandialglucoselevelsindiabeticpatients[4].Metforminremainsago
odfirst-
linepharmacologictreatmentfortype2diabetesinmostpatientsandusefulintreatmentofpol ycysticovarysyndrome[5,6].Metforminimproveingovarianangiogenesis,reducecystfor mationinpolycysticovarysyndromeratmodelandimprovingfolliculardevelopmentandov ulation[7].Repaglinideandmetformincancompensatethedamagecausedbypolycysticova riantissu[8].
METHODS
FemalewhiteadultalbinoratsobtainedfromtheBabylonUniversityanimalhouseoftheBiol ogyDepartment/collegeofScience,agedbetween2-3monthsandweighingabout150–
170gwereusedinthisstudy.Thefemaledividedintotwogroupsthefirstgroupwereusedforth einductionofdiabetesmellitustypeІІusingalloxanmonohydratetofastedratsbyasingledose ofalloxan130mg/kgbodyweightintraperitonealy[9]after72hofinjection,fastingbloodglu coselevelwasmeasuredafterstarvationfor9-
12houroffastingratsusingtheglucometer.Theratswithcontinualbloodglucoselevelabove 200mg/dlforsevendaywereconsideredtobehyperglycemicandwereselectedfortreatment, thesecondgroupoffemaleratweregivennormalsalineintraperitonialy,bothtwofemalegrou psweredividedintothreesubgroupsthatincluded.Group1Normalcontrol(NC),Group2No n-diabeticratstreatedwithrepaglinide(N+Rep)4mg/kg/,Group3Non-
diabeticratstreatedwithmetformin(N+Met)1000mg/kg/day,Group4Diabeticcontrol(DC )includedanimalswhichhadbeengivenalloxani.p.andadministrateddistilledwaterorallyb utdidnottreatedwithdrugs,Groups5Diabeticratswithrepaglinid(D+Rep)whichreceivedal loxani.p.andaftertheinductionofDMbysevendays,thediabeticratstreatedwithrepaglinide 4mg/kg/dayandGroups6Diabeticratswithmetformin(D+Met)whichreceivedalloxani.p.a ndaftertheinductionofDMbysevendaysthediabeticratstreatedwithmetformin1000mg/kg /day.
Vaginalsmearwastakendailytodeterminethestageoftheoestrouscycleanddeterminethedu rationofoestrouscycleforallgroups,byobtainedvaginalsmearsamples,Thenair-
driedthesmearsamples,stainedthemtoexaminebymicroscopyandnotedcellpredominance todetermineestrouscyclephase[10].
Estrouscyclephasewasrecordedforeachratasestrous,diestrousorproestrous.After6weeks oftreatment,apreponderanceofuniformlynucleatedcellsindicatingtheproestrusphaselate ratthenextdaytheratsweresacrificedbyventrallaparotomyalsotheovarieswerecollectedan dexcessfattissueswereremovedandthenweighted.
Subsequently,histologicalsectionsofoverieswerepreparedtoinvestigatethemeantotalnu mberoffolliclesandcorpusluteaincontrolandtreatmentgroupstoevaluatetheeffectoftreat mentonovarianfunction.
RESULTS
Theresultsofovarianweightrevealedsignificantdecrease(p≤0.05)inDCascomparedtoNC groupbutsignificantincreaseinD+RepgroupascomparedtoDCgroup.Whereas,non-
significantdifference(p≤0.05)inN+RepgroupandN+Metgroupwhencomparedtonormalc ontrolgroup.
Theovariansectionstakenfromthenormalcontrolanimalsgroupshowedanaturalstructure withfolliclesatdifferentstagesofgrowth.Numberofprimaryfolliclesresultsexposedthere wasnon-significantdecrease(p≤0.05)inDCgroupascomparedtoNCgroup,butnon- significantdifferences(p≤0.05)inD+RepgroupandD+MetgroupascomparedtoDCgroup.
Alsonon-
significantdifferences(p≤0.05)inprimaryfolliclesnumberinN+RepgroupandN+Metwhe ncomparedtoNCgroup.
Asexposedintable(1)secondaryfolliclesnumberresultsrevealedsignificantincrease(p≤0.
05)insecondaryfolliclesnumberinN+MetgroupascomparedtoNCgroupbutnon- significantincrease(p≤0.05)inN+RepgroupascomparedtoNCgroup.
Resultsofcorpusluteumnumberinexperimentalgroupexposedtherewassignificantdecrea se(p≤0.05)inDCgroupascomparedtoNCgroupbutnon-
significantdifference(p≤0.05)amongD+RepgroupandD+MetgroupascomparedtoDCgr oupandNCgroup.Therewassignificantincrease(p≤0.05)inN+MetgroupascomparedtoN CgrouphoweverN+Repgrouprevealednon-
significantdifference(p≤0.05)ascomparedtoNCgroup.
Thefolliclesandcorpusluteacountsexposedtherewassignificantdecrease(p≤0.05)inDCgr oupascomparedtoNCgroup.butnon-
significantdifferences(p≤0.05)inD+RepgroupascomparedtoDCgroupandNCgroup.The resultofD+Metgroupshowedsignificantincrease(p≤0.05)ascomparedtoDCgroup.There wassignificantincrease(p≤0.05)inN+MetgroupascomparedtoNCgroup.
OvarianFollicleandCorpusLuteumCountsinExperimentalVirginFemaleAlbinoRa ts.
Groups
Parameters NC (mean±
S.E)
N+Rep (mean±
S.E)
N+Met (mean±
S.E)
DC(me an±S.E )
D+Rep(
mean±
S.E)
D+Met(
mean±
S.E)
Pvalu e
Weightofov ary
0.09bc
±0.007
0.100c
±0.009
0.08bc
±0.004
0.05a
±0.006
0.08bc
±0.004
0.07ab
±0.004
<0.00 1 numberofpr
imaryfollicl es
7.66ab
±0.9
9.16b
±2.25
6.16ab
±1.13
4.33a
±0.49
5.00a
±0.85
5.00a
±0.68 0.06 numberofse
condaryfolli cles
15.16ab
±1.40
23.00bc
±2.17
26.33c
±5.03
12.33a
±1.25
15.16ab
±1.68
21.00abc
±3.40 0.01 numberofco
rpusluteum
16.66b
±1.28
15.50ab
±1.23
20.66c
±2.01
11.66a
±0.66
14.00ab
±1.15
13.33ab
±0.66
<0.00 1
A B
D
E F
c
Numberoffo lliclesandco rpuslutea
39.50bc
±2.01
47.66cd
±4.35
53.16d
±5.23
28.32a
±1.54
34.16ab
±1.93
39.33bc
±4.35
<0.00 1
Figure (1) Section through the ovary showing different stages of maturation of follicles with the appearance of corpus luteum (CL). H&E. (A and B) primary follicle X100 ,(C and D) Secondary Follicle,( E) Secondary Follicle (antral) and(F) Secondary Follicle (Graafian Follicle).
Figure (2) Section through the ovary showing different stages of maturation of follicles with the appearance of corpus luteum (CL). H&E.
DISCUSSION
Oneoftheessentialindexesintheassessmentofovarianfunctionsisthealterationintheweight andsizeofovary,whichiscausedalterationinovarianstructures,likethechangeinthenumber andnatureoffollicles[11]Ovarianweightresultsinpresentstudyrevealedsignificantdecrea seinovarianweightof(DC)groupascomparedto(NC)groupthispossiblybecausehyperglyc
emiainblood causes
reductionofbodyweight,decreasesthenumberofdynamicfolliclesandincreasesatreticfolli cleshasdestructiveeffectsonovariantissueresultinreducedweightandsizeofovaryinSTZ- induceddiabetes[11].
Treatmentwithrepaglinidein(D+Rep)grouprevealedsignificantimprovementinovarianw eightascomparedto(DC)groupthisispossiblybecauserepaglinideloweringbloodglucosel evelintype2diabetesmellitusPatientsThusrepaglinidereducesdiabetescomplications[8,1 2].
Numberofprimaryfolliclesresultsinpresentstudyrevealedthat(N+Met)grouphasthelarge stnumberandtherewasnon-
significantdifferencesamongallexperimentalgroupsthisresultsagreewith[13]Resultsofse condaryfolliclesnumberexposedsignificantincreasedin(N+Rep)and(N+Met)groupsthis maybebecauseMetforminimproveingovarianfollicledynamicsbyimprovesthecarbohydr atemetabolism[14]ormaybebecausethesetreatmentcausingovarianstimulation.Thisresul tapprovewith[11].Also,theuseofinsulin-
sensitizingagentsasMetforminandRipaglinidetotreatmentthePolycysticovarysyndrome exposedthesedrugscandevelopfolliculargrowthandcompensatethedamagesduetoPolycy sticovarysyndrome[8]. Also recent in vitro study of [21] revealed that supplementation of the culture medium with repaglinide can improve the ovarian follicle survival, growth and subsequently in vitro oocyte maturation..
Numberofcorpusluteumresultsofpresentstudyexposedthereissignificantdecreasein(DC) groupascomparedto(NC)groupthisresultpossiblybecausediabetesreducedovulation[15].
HyperglycemiacausessevereovarianinjuryleadingtothedecreaseinovarianreserveinSTZ -induceddiabeticrats[16].Thisresultagreewith[17].
Normalwithmetformingrouprevealssignificantincreaseincorpusluteumnumbers,Metfor minimprovingfolliculardevelopmentandthepercentageofcorporaluteainafemalepolycys ticovarysyndromeratmodel [7]
Toassessedtheovarianfunctionmustcomparetheovarianfollicletocorpusluteumcounts [18].Theresultsofpresentstudyexposethereissignificantrisingintotalnumberoffolliclesan dcorpusluteaofN+MetgroupwhencomparedwithNCgroupandissignificantrisinginD+M etascomparedtoDCgroupthisindicatesthatmetforminhasapositiveeffectonimprovingova rianfunction.ThismaybebecauseMetforminactsindirectlytoimproveovulationandreduce long-termmetabolic
complications[19].Diabetesmellituscausedsignificantdecreasingincorporaluteaandovar ianfolliclesnumbersDCgrouppossiblybecauseHyperglycemicausesovarianstromalandf olliculardegenerationthenfibrosis,Moreover,metforminexposedbeneficialeffectsinredu cingthesecomplications [20].
CONCLUSION
Diabetes mellitus causes decrease in ovarian weight,number of secondary follicles and number of corpus luteum. treatment with repaglinide result in improvement of ovarian weight and treatment with metformin result in improvement number of follicles and corpus lutea.REFERENCES
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