Original Article
Nitric Oxide-Induced Polycystic Ovaries in The Wistar Rat Fatemeh Hassani, M.Sc.1, Manizheh Karami, Ph.D. 1*, Mohammad Reza Jalali Nadoushan, Ph.D.2, Poopak Eftekhari Yazdi, Ph.D.3 'HSDUWPHQWRI%LRORJ\)DFXOW\RI%DVLF6FLHQFHV6KDKHG8QLYHUVLW\7HKUDQ,UDQ 'HSDUWPHQWRI3DWKRORJ\6FKRRORI0HGLFLQH6KDKHG8QLYHUVLW\7HKUDQ,UDQ 'HSDUWPHQWRI(PEU\RORJ\5HSURGXFWLYH%LRPHGLFLQH5HVHDUFK&HQWHU5R\DQ,QVWLWXWH IRU5HSURGXFWLYH%LRPHGLFLQH$&(&57HKUDQ,UDQ
Abstract
%DFNJURXQG 1LWULFR[LGH 12 LQYROYHVLQSRO\F\VWLFRYDU\V\QGURPH 3&26 DFDXVH RILQIHUWLOLW\LQZRPHQGXULQJWKHUHSURGXFWLYHDJH7KH3&26LVQRZFDWHJRUL]HGDVDQ LQÀDPPDWRU\SKHQRPHQRQ7KHDLPRIWKLVVWXG\ZDVWRHYDOXDWHWKHUROHRI12DSUR LQÀDPPDWRU\DJHQWLQWKLVV\QGURPHDWKLVWRORJLFDODQGELRFKHPLFDOOHYHOV 0DWHULDOV DQG 0HWKRGV In this experimental study, animals were female Wistar rats ZHLJKLQJJ NHSWXQGHUVWDQGDUGFRQGLWLRQV/$UJLQLQH PJNJ DSUHFXUVRURI12ZDVLQMHFWHGLQWUDSHULWRQHDOO\ LS WKURXJKDSHULRGUDQJLQJIURP WRGD\V once a day. The rats' estrous cycle was studied using Papanicolaou test; those showing phase RI'LHVWURXVZHUHJURXSHGLQWRH[SHULPHQWDODQGFRQWUROJURXSV7KHFRQWUROJURXSVROHO\ UHFHLYHGVDOLQH PONJLS WKURXJKRXWDOOH[SHULPHQWV7RHYDOXDWHWKHLQÀDPPDWRU\HIIHFW RI12WKHUDWVZHUHWUHDWHGDQDQWLLQÀDPPDWRU\DJHQWQDOR[RQHK\GURFKORULGH PJNJ LS SULRUWR/DUJLQLQH$WWKHHQGRIWKHWUHDWPHQWSHULRGDOODQLPDOs’RYDULHVZHUHDVVHVVHG IRUKLVWRSDWKRORJLFDODQGKLVWRFKHPLFDOLQYHVWLJDWLRQV$OVRDFWLYDWLRQRI12V\QWKDVH 126 LQWKHH[SHULPHQWVZDVVWXGLHGXVLQJ1$'3+GLDSKRUDVHWHFKQLTXH
5HVXOWV 7KHRYDULHVRIUDWVWUHDWHGZLWK/DUJLQLQHVKRZHGSRO\F\VWLFFKDUDFWHULVWLFVLQ contrast to those collected from control or naloxone pretreated groups, based on image anal\VLV$GLIIHUHQFHLQHQ]\PHDFWLYDWLRQZDVDOVRVKRZQLQWKHVHFWLRQVWKDWEHORQJHGWRWKH JURXSVWKDWUHFHLYHG/DUJLQLQHZKHQFRPSDUHGZLWKWKHSUHQDOR[RQHDQGFRQWUROJURXSV &RQFOXVLRQ %DVHGRQWKHVHUHVXOWVZHEHOLHYHWKDW12PD\SOD\DPDMRUUROHLQWKH SDWKRSK\VLRORJ\RI3&26 Keywords:3&261LWULFR[LGH/$UJLQLQH1DOR[RQH1$'3+'LDSKRUDVH Citation: Hassani F, Karami M, Jalali Nadoushan MR, Eftekhari Yazdi P. Nitric oxide-induced polycystic ovaries in the wistar rat. Int J Fertil Steril. 2012; 6(2): 111-116.
Introduction 3RO\F\VWLFRYDULDQV\QGURPH 3&26 LVLQWURGXFHG DVFRPPRQHQGRFULQHGLVRUGHUWKDWDIIHFWVXSWR RI ZRPHQ GXULQJ WKHLU UHSURGXFWLYH DJHV 7KLV disoreder is manifested by heterogeneous clinical feaWXUHV DVLWFKDUDFWHUL]HGE\KLUVXWLVPLUUHJXODU PHQVWUXDO F\FOHV DQG LQIHUWLOLW\ ,Q DQRYXODWRU\ ZRPHQVXIIHULQJIURP3&26WKHSURPLQHQWRYDULDQ VLJQVDUHOLVWHGDVWKHIROOLFXODUPDWXUDWLRQDUUHVW
Received: 14 Jun 2011 , Accepted: 5 Feb 2012 * Corresponding Address: P.O.Box: 18155/159, Deptartment of Biology, Faculty of Basic Sciences, Shahed University, Tehran, Iran Email: [email protected]
DQG LQVXOLQ UHVLVWDQFH 7KH ODWHU SKHQRPHQRQhas EHHQUHODWHGZLWKLPSDLUHGSURGXFWLRQUHOHDVHRI HQGRWKHOLXPGHULYHGQLWULFR[LGH 12 DQGLQFUHDVHG OHYHOVRIHQGRWKHOLQNQRZQDVPDLQPDUNHUVRIYDVFXODUGLVHDVH NO is indicated as an important paracrine mesVHQJHUWKDWSDUWLFLSDWHVLQVHYHUDOSK\VLRORJLFDODQG SDWKRSK\VLRORJLFDOHYHQWVLQWKHHQGRFULQHRUJDQV This molecule is a free-radical produced from the oxi-
Royan Institute International Journal of Fertility and Sterility Vol 6, No 2, Jul-Sep 2012, Pages: 111-116
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dation of terminal guanidino nitrogen of arginine by WKHDFWLRQRIQLWULFR[LGHV\QWKDVH HQ]\PH 126 7KHPROHFXOHLVZHOOGRFXPHQWHGDVDORFDO inflammatory generator and included with the factors introduced as those responsible for the RYXODWRU\ SURFHVVHV DQG WKH 3&2 V\QGURPH DV ZHOO 1DOR[RQHDQDUFRWLFGUXJLVXVHGDVD QRYHODQWLLQÀDPPDWRU\DJHQW 7KLV VWXG\ HYDOXDWHV WKH LQIOXHQFH RI /DUJLQLQH D SUHFXUVRU RI 12 LQ 3&26LQGXFWLRQ LQIHPDOH:LVWDUUDWV1$'3+GLDSKRUDVHSURYLGHV YDOXDEOH ELRFKHPLFDO GDWD UHSUHVHQWLQJ the participation of NO in this phenomenon. The marker is classified as the best indicator RI 126 HQ]\PH :H KDYH DOVR H[DPLQHG WKH metabolic status in L-arginine-exposed rats and HYDOXDWHGWKHHIIHFWRIQDOR[RQHLQ/DUJLQLQH treated rats.
Materials and Methods Animals In this experimental study, the animals were eight-week-old female Wistar rats (weight: J SXUFKDVHG IURP 3DVWHXU ,QVWLWXWH RI ,UDQ 7HKUDQ ,UDQ $QLPDOV ZHUH UHWDLQHG under standard conditions in accordance to the *XLGHIRUWKH&DUHDQG8VHRI/DERUDWRU\$QLPDOV $QLPDOV ZHUH PDLQWDLQHG LQ D VWDQGDUGWHPSHUDWXUH Û& DQGKRXUVOLJKW dark cycle with food and water ad libitum. All H[SHULPHQWVZHUHDSSURYHGE\WKHORFDO(WKLFDO &RPPLWWHHDW6KDKHG8QLYHUVLW\ Drugs /DUJLQLQH QLWUREOXH WHWUD]ROLXP 1%76 DQG ȕQLFRWLQDPLGH DGHQLQH GLQXFOHRWLGH SKRVSKDWH ȕ1$'3+ ZHUHSXUFKDVHGIURP6LJPD&KHPLFDO &R 6W /RXLV 02 86$ 1DOR[RQH K\GURFKORULGHZDVSURYLGHGE\WKH7ROLG'DUX&R7HKUDQ ,UDQ .HWDPLQH DQG [\OD]LQH ZHUH REWDLQHG IURP9HWHULQDU\2UJDQL]DWLRQ7HKUDQ,UDQ Papanicolaou (PAP) stain )HPDOH UDWV KDYH D GD\ F\FOH LQFOXGLQJ proestrous (12 hours follicular growth and peak esWURJHQ SKDVH HVWURXV KRXUV RYXODWLRQ SKDVH metestrous (12 hours corpus luteum secrets progesWHURQH SKDVH DQG GLHVWURXV KRXUV FRUSXV IJFS, Vol 6, No 2, Jul-Sep 2012
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OXWHXPUHJUHVVLRQSKDVH :LWKRXWH[SRVLQJZLWK male rats, the female rats are always in the diestrous phase; their phases will progress only DIWHU PDWLQJ ZLWK D PDOH UDW :LWK UHJDUGV WR WKH DERYHPHQWLRQHG H[SODQDWLRQ ZH FKRVH YLUJLQ UDWV LQ RXU VWXG\ %HFDXVH YDJLQDO changes may appear through the experiments, the animals’YDJLQDOVPHDUVZHUHDOVRH[DPLQHG during the entire experiment by the Papanicolaou 3$3 VWDLQ,QWKLVSURFHGXUHWKHVPHDUVREWDLQHG E\YDJLQDOZDVKLQJEHWZHHQ DPDQG pm were stained using a PAP stain for humans, DVGHYHORSHGLQSUHYLRXVVWXGLHV Three W\SHVRIFHOOVZHUHUHFRJQL]HGLQWKH3$3VWDLQHG smears: i. round and nucleated epithelial cells;ii. LUUHJXODUFRUQL¿HGFHOOV LLLOLWWOHURXQGFHOOV OHXNRF\WHV 7KH FHOO SURSRUWLRQ LQ WKH VPHDU ZDV used to determine the estrous cycle phase as preYLRXVO\EHHQSURSRVHG Drug administrations )HPDOH:LVWDUUDWV J DWHLJKWZHHNVRI DJHZHUHUDQGRPO\GLYLGHGLQWRWKH/DUJLQLQHGRVH JURXSV PJNJ Q LQ HDFK DQG D JURXS WKDW ZDV SUHLQMHFWHG ZLWK QDOR[RQH PJNJ Q SULRUWR/DUJLQLQHDGPLQLVWUDWLRQ7KHGUXJ /DUJLQLQHZDVLQMHFWHGLQWUDSHULWRQHDOO\ LS RQFH GDLO\GXULQJDSHULRGWKDWUDQJHGIURP WRGD\V 1DOR[RQHZDVLQMHFWHGRQFHSHUGD\PLQXWHVSULRU to L-arginine administration. The control group reFHLYHGVDOLQH PONJLS LQVWHDGRIWKHGUXJV Surgery procedure for removal of ovaries 7KHWUHDWPHQWJURXSVZHUHDQHVWKHWL]HGE\GLHWK\OHWKHULQDVSHFLDOGHYLFH7KHQDFPPLGOLQH incision in the lower abdomen area was performed. 7KHRYDULHVZHUHFDUHIXOO\H[DPLQHGELRPHWULFDOO\DQGGLVVHFWHGRXWWKHQFROOHFWHGLQD¿[DWLYHIRU further histological and biochemical analysis. +LVWRORJLFDODQDO\VLV )RUKLVWRORJLFDOLQYHVWLJDWLRQVWKHRYDULHVZHUH ¿[HG LQ D IRUPDOLQ VROXWLRQ DQG SURFHVVHG ZLWK D WLVVXH SURFHVVRU WKURXJK SDUDI¿Q HPEHGGLQJ6HULDOVHFWLRQV P ZHUHSUHSDUHGZLWK a rotary microtome. The slides were then stained XVLQJ WKH KHPDWR[\OLQ DQG HRVLQ + ( VWDLQLQJ PHWKRG DQGFOHDUHGZLWK[\OHQH$IWHUPRXQWLQJWKHVOLGHVZHUHHYDOXDWHGZLWKDOLJKWYLGHR SKRWRPLFURVFRSH 2O\PSXV 86$ DQG D YLGHR
PCOS Model
SKRWRELQRFXODUDWWKHGHVLUHGPDJQL¿FDWLRQ +RUPRQHDVVD\V Blood sampling for serum parameters At the end of the experiment, the rats were anesWKHWL]HG E\ LS LQMHFWLRQV RI PJNJ NHWDPLQH K\GURFKORULGH DQG PJNJ [\OD]LQH +HDUW EORRG VDPSOHVZHUHREWDLQHGEHWZHHQ DPDIWHU DQRYHUQLJKWIDVWWRDVVHVVVXJDUOLSLGDQGKRUPRQDO SUR¿OHV$OOVDPSOHVZHUHNHSWDWURRPWHPSHUDWXUH IRUDWOHDVWPLQXWHVWRIDFLOLWDWHWKHFORWWLQJRIWKH VDPSOHV7KHVDPSOHVZHUHWKHQFHQWULIXJHGDW JIRUPLQXWHV7KHVHUDZHUHVWRUHGDWíÛ& IRU IXWXUH DVVHVVPHQWV 7KH OHYHO RI VHUD DJHQWV >KLJKGHQVLW\ OLSRSURWHLQ +'/ ORZGHQVLW\ OLSRSURWHLQ /'/ JOXFRVH FKROHVWHURO DQG WULJO\FHUides] were measured based on competition binding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Û& IROORZHG E\ FU\RSURWHFWLRQLQVXFURVH7KHQVSHFLPHQVZHUHVHFWLRQHG ZLWK FU\RVWDW PLFURWRPH DW Û& P IUR]HQ VHFWLRQV DQGPRXQWHGRQSRO\/O\VLQHFRDWHGVOLGHV 7KHVOLGHVZHUHULQVHGZLWKEXIIHUDQGWKHQ stained using the nicotinamide adenine dinucleotide SKRVSKDWH 1$'3+ GLDSKRUDVH WHFKQLTXH WR YLVXDOL]H126DFWLYLW\%ULHÀ\WKHSUHSDUHGVOLGHVZHUH LQFXEDWHGZLWKVKDNLQJVWDWHLQD7ULWRQ; LQSKRVSKDWHEXIIHUIRUWRPLQXWH V 7KHVWDLQing was then performed by incubating the slides in a solution containing equal parts of nitro-blue tetra]ROLXP 1%7PJPOLQEXIIHU DQG1$'3+ PJPOLQEXIIHU IRUDERXWKRXUVDWÛ&8SRQ UHGXFWLRQE\1$'3+GLDSKRUDVH1%7\LHOGVDEOXH IRUPD]DQWKDWLVYLVLEOHE\OLJKWPLFURVFRS\&RQWURO specimens were assessed using the same procedure except that the specimens were placed in an incubaWLRQEDWKGHYRLGRIWKHPDUNHU1RUHDFWLRQWR1$'3+ZDVREVHUYHGLQWKHFRQWUROVDPSOHV7KHWLVVXH samples were then dehydrated in ascending series of
ethanol, cleared in xylene alcohol and xylene, and PRXQWHGLQ(QWHOODQ 0HUFNFR*HUPDQ\ Image analysis 7KHWLVVXHVOLGHVZHUHREVHUYHGXQGHUWKHYLGHRSKRWROLJKWPLFURFRSH 2O\PSXV86$DQGWKHSURYLGHGLPDJHVZHUHWKHQDVVHVVHGLQP2 units of the photorecords at î RU PRUH PDJQL¿FDWLRQ XVLQJ WKH ,PDJH 7RRO SURJUDP 87+6&6$ YHUVLRQ WKHIUHHLPDJHSURFHVVLQJDQGDQDO\VLV SURJUDPIRU0LFURVRIW:LQGRZVWRSURYLGHTXDQWL¿FDWLRQIRUDQDUHDRIP2. Statistical analysis 'DWDZHUHDQDO\]HGE\WKH.ROPRJRURY±6PLUQRY .6 WHVWIRUVKRZLQJWKHHTXDOLW\WRDQDO\VLVE\ YDULDQFH $129$ XVLQJ6366VRIWZDUH YHUVLRQ 6366,QF&KLFDJR,/ $7XNH\.UDPHURU /6'SRVWKRFWHVWZDVXVHGWRFDOFXODWHWKHGLIIHUHQFHEHWZHHQWKHJURXSVDIWHU$129$6WDWLVWLFDO VLJQL¿FDQFHZDVFRQVLGHUHGS $OOGDWDZHUH H[SUHVVHGDVPHDQ6(0,QFDVHRIWKH1$'3+ diaphorase technique, the intensity analysis was DVVHVVHGDWP2 using the Image Tool program 87+6&6$YHUVLRQ DIWHUFDOLEUDWLQJIRUD P2 area.
Results Papanicolaou (PAP) stain 7KHHVWURXVF\FOHSKDVHRIIHPDOHYLUJLQUDWVZDV determined as diestrous after using the PAP stain )LJ 5RXQGQXFOHDWHG HSLWKHOLDO FHOOVZHUHWKH PRVWDEXQGDQWFHOOW\SHREVHUYHGLQWKHVPHDUV.
Fig 1: Result of Papanicolaou (PAP) stain of female virgin rat vagiQDOVPHDUVLQWKHGLHVWURXVSKDVH7KHDEXQGDQFHRIOHXNRF\WHVLQ WKHVPHDUVDPSOHVLQGLFDWHWKDWWKHF\FOHSKDVHLVGLHVWURXV
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+LVWRORJ\ The rats’ RYDULHV IURP WKH /DUJLQLQH GRVH JURXSV PJNJ H[KLELWHG VPDOO IROOLFOHVLQWKHHDUO\GHYHORSPHQWSKDVHLQDGGLWLRQ WR WKH IROOLFOHV WKDW VKRZHG HYLGHQFH RI HLWKHU atresia, large cysts with thickened granulosa cell layer, or large cystic follicles with scant JUDQXORVDFHOOV )LJ% 7KHVDPSOHVRIWKRVH SUHDGPLQLVWHUHGZLWKQDOR[RQH PJNJ )LJ & VKRZHGDUHGXFWLRQLQWKHLQFLGHQFHRIF\VW formation. The control group exhibited a follicular appearance depending on the phase of the cycle; in diestrous, only secondary follicles and fresh corpora lutea were seen. Atretic folOLFOHV ZHUH DOVR REVHUYHG LQ WKH SKDVHV RI WKH control group. The corpus luteum was absent in DOOFDVHV )LJ$
B
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Fig 2: Pictures of ovaries from control (a, A), solely L-arginineWUHDWHG % DQGQDOR[RQHSUHWUHDWHG & UDWV%DUVEHVLGHWKH VDPSOHVVKRZPPYDOXHV7KH¿JXUHVDUHVKRZQXQGHUPDJQL¿FDWLRQ>D îREMHFWLYHRIDQ LQYHUWHG SKRWRPLFURVFRSH $ %& 2O\PSXVSKRWRPLFURVFRSHDWî@
1$'3+GLDSKRUDVHUHDFWLYLW\ 3RVLWLYH 1$'3+GLDSKRUDVH UHDFWLYLW\ ZDV REVHUYHGLQWKHVWURPDDQGLQWKHWKHFDFHOOOD\HULQWKH RYDULHVRI/DUJLQLQHWUHDWHGUDWV )LJ$ 7KHUHDFWLYLW\ZDVDOVRREVHUYHGLQWKHPHPEUDQHJUDQXORVD FHOODUHDRIWKHIROOLFOHVLQWKHVDPSOHV,QOXWHLQL]HG RYDULHV ZHDNHU GLDSKRUDVH UHDFWLYLW\ ZDV REVHUYHG in patches at the periphery of the corpora lutea. The UHVSRQVHWR1$'3+GLDSKRUDVHVKRZHGDWWHQXDWLRQ LQQDOR[RQHSUHLQMHFWHGVDPSOHV )LJ% ,QDFRPSDULVRQDQDO\VLVFRPSOHWHGE\,PDJHWRROQRVLJQL¿FDQW UHDFWLRQ ZDV REVHUYHG LQ WKH FRQWURO VSHFLPHQ )LJ& ,WVKRXOGEHQRWHGWKDWWKHDUHDVRILQWHUHVW FRQWDLQHG 12JHQHUDWLQJ HQ]\PH DFWLYLW\ DQG WKDW WKHDFWLYLW\RI126ZDVLQDJUHHPHQWZLWKWKHWLPHRI rupture and cystic formation.
A
IJFS, Vol 6, No 2, Jul-Sep 2012
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PCOS Model
Blood Sugar
Cholesterol
Triglyceride
HDL
LDL
Level in serum (mg/dl)
200
100
0 Control
A
L-Arg
Nal+L-Arg
Tratment
Fig 4: Curves indicate the levels of metabolic agents in seUXP PJG/ ERWK LQ WKH FRQWURO DQG H[SHULPHQWDO JURXSV Q 7KHH[SHULPHQWDOUDWVUHFHLYHG/DUJLQLQH PJ NJLSGD\V RU/DUJLQLQH PJNJLSGD\V SOXV QDOR[RQH PJNJLSGD\V 1DOR[RQHZDVLQMHFWHG PLQXWHVSULRUWR/DUJLQLQH9DOXHVDUHPHDQ6(0
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Discussion
B
C Fig 3: +LVWRFKHPLFDO1$'3+GLDSKRUDVHHYDOXDWHGLQFRQWURO (A) and treated rats; single L-arginine (B) or L-arginine + naloxRQH & DVGHWDLOHGLQPDWHULDOVDQGPHWKRGV
Serum agents levels /HYHOVRIVHUXPSDUDPHWHUVDUHVKRZQLQ)LJ$V the data denotes, L-arginine treatment induced a sigQL¿FDQWFKDQJHLQWKHOHYHORI/'/LQFRQWUDVWZLWK WKHFRQWURO7KHQDOR[RQHJURXSVKRZHGQRVLJQL¿cant difference to the control, meaning that the agent LQWHJUDWHGWKHGHYLDWLRQOHYHO )LJ
12LVFDWHJRUL]HGDVDQLPSRUWDQWLQWUDRYDULDQPHGLDWRU WKDW DIIHFWV WKH RYXODWRU\ SURFHVV DQG regulates the functions of the corpus luteum. The present study has lent further support for a functional role RI12LQWKHRYDULDQIXQFWLRQV$FWLYDWLRQRIWKH126 HQ]\PHZDVGHPRQVWUDWHGXVLQJ1$'3+GLDSKRUDVH 7KHSUHVHQW¿QGLQJVVKRZHGWKHDFWLYLW\RIWKLVHQ]\PH ERWKLQWKHVWURPDODQGWKHFDOOD\HUVLQRYDULHVWUHDWHG with L-arginine. 7KLV VWXG\ SURYLGHG PRUH HYLGHQFH IRU IROOLFXODU atresia, as well as the production of large cysts due to 12WUHDWPHQWLQ:LVWDUUDWVZKLFKDJUHHGZLWKDSUHYLRXV¿QGLQJ'HVSLWHWKHYDVFXODUHIIHFWRI12ZKLFK IRUPVWKHKLJKO\UHDFWLYHF\WRWR[LFSHUR[\QLWULWH LW PD\PHGLDWHWKHFHOOXODUF\WRWR[LFLW\RIRYDULHVE\WKH 3&26SKHQRW\SHHIIHFWDQGYLDF\WRWR[LFSHUR[\QLWULWH NO production and formation of the free radicals may KDYH VRPH LPSRUWDQFH LQ WKH RYXODWRU\ SURFHVV 7KLV IDFWPD\IDFLOLWDWHWKHFDWLVVXHHYHQWV7KHWXQLFDDOEXJinea of the follicle wall may be one of the symptom’s VLJQVDVEHHQQRWHGSUHYLRXVO\ ,QWKHSUHVHQWVWXG\ WKHVHDUHDVKDYHEHHQIRXQGWRFRQWDLQ12JHQHUDWLQJ HQ]\PHDFWLYLW\DQGWKH126DFWLYLW\DJUHHGZLWKWKH time of rupture and cystic formation. ([FHSWIRUWKHOHYHORIVHUXP/'/ )LJ QRVLJQL¿FDQW FKDQJH LQ RWKHU OHYHOV VXFK DV JOXFRVH RU EORRG VXJDUZDVIRXQG1DFXOHWDO VKRZHGDQHJDWLYH correlation between NO and markers of glucose hoPHRVWDVLVLQ3&26SDWLHQWV7KH12OHYHOVDSSHDUHGWR
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be regulated by endogenous estrogens because a higher OHYHOZDVGHVFULEHGLQWKHIROOLFXODUSKDVHWKDQLQWKH OXWHDOSKDVHRIWKHPHQVWUXDOF\FOH 7KHVHGDWDSRVVLEO\ VXJJHVW WKDW WKH HVWURJHQ OHYHOV LQ WKH GLHVWURXV phase may interfere with NO secretion, which is preVXPDEO\UHODWHGWRWKHSUHVHQFHRI3&26 7KRXJKQRVLJQL¿FDQWFKDQJHLQVHUDOHYHOVZDVIRXQG the administration of naloxone prior to L-arginine led to D GHFUHDVH LQ WKH OHYHO RI VHUXP /'/ )LJ DQG DWWHQXDWHG12SURGXFLQJDFWLYLW\ )LJ 7KHVH¿QGLQJV possibly demonstrate that the NO molecule, as a proLQÀDPPDWRU\HOHPHQWPD\LQGXFH3&26E\DFWLYDWLQJ LQÀDPPDWRU\IDFWRUVLQRYDULHVDQGWKHDQWLLQÀDPPDtory agent naloxone affects the endocrine and metabolic PHDVXUHVUHOHYDQWWR3&261DOWUH[RQHDGPLQLVWUDWLRQ has been shown to result in reduced food intake in anGURLG REHVLW\ 0HWDEROLF DEQRUPDOLWLHV VXFK DV hyperinsulinemia, insulin-resistance, and obesity, are OLVWHG DV FRPPRQ IHDWXUHV RI 3&26$ OLQN EHWZHHQ RSLRLGVDQG3&26UHODWHGLQVXOLQUHVSRQVHWRJOXFRVH ORDGKDVDOVREHHQSUHYLRXVO\VXJJHVWHG
Conclusion 7KH¿QGLQJVVKRZDFRUUHODWLRQEHWZHHQFHQWUDORSLoid tone and body weight. The regulation of metabolic agents by naloxone in this study beyond the effect of the GUXJRQ3&26VLJQVUHPDLQVHOXVLYH
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Nitric Oxide-Induced Polycystic Ovaries in The Wistar Rat Fatemeh Hassani, M.Sc.1, Manizheh Karami, Ph.D. 1*, Mohammad Reza Jalali Nadoushan, Ph.D.2, Poopak Eftekhari Yazdi, Ph.D.3 'HSDUWPHQWRI%LRORJ\)DFXOW\RI%DVLF6FLHQFHV6KDKHG8QLYHUVLW\7HKUDQ,UDQ 'HSDUWPHQWRI3DWKRORJ\6FKRRORI0HGLFLQH6KDKHG8QLYHUVLW\7HKUDQ,UDQ 'HSDUWPHQWRI(PEU\RORJ\5HSURGXFWLYH%LRPHGLFLQH5HVHDUFK&HQWHU5R\DQ,QVWLWXWH IRU5HSURGXFWLYH%LRPHGLFLQH$&(&57HKUDQ,UDQ
Abstract
%DFNJURXQG 1LWULFR[LGH 12 LQYROYHVLQSRO\F\VWLFRYDU\V\QGURPH 3&26 DFDXVH RILQIHUWLOLW\LQZRPHQGXULQJWKHUHSURGXFWLYHDJH7KH3&26LVQRZFDWHJRUL]HGDVDQ LQÀDPPDWRU\SKHQRPHQRQ7KHDLPRIWKLVVWXG\ZDVWRHYDOXDWHWKHUROHRI12DSUR LQÀDPPDWRU\DJHQWLQWKLVV\QGURPHDWKLVWRORJLFDODQGELRFKHPLFDOOHYHOV 0DWHULDOV DQG 0HWKRGV In this experimental study, animals were female Wistar rats ZHLJKLQJJ NHSWXQGHUVWDQGDUGFRQGLWLRQV/$UJLQLQH PJNJ DSUHFXUVRURI12ZDVLQMHFWHGLQWUDSHULWRQHDOO\ LS WKURXJKDSHULRGUDQJLQJIURP WRGD\V once a day. The rats' estrous cycle was studied using Papanicolaou test; those showing phase RI'LHVWURXVZHUHJURXSHGLQWRH[SHULPHQWDODQGFRQWUROJURXSV7KHFRQWUROJURXSVROHO\ UHFHLYHGVDOLQH PONJLS WKURXJKRXWDOOH[SHULPHQWV7RHYDOXDWHWKHLQÀDPPDWRU\HIIHFW RI12WKHUDWVZHUHWUHDWHGDQDQWLLQÀDPPDWRU\DJHQWQDOR[RQHK\GURFKORULGH PJNJ LS SULRUWR/DUJLQLQH$WWKHHQGRIWKHWUHDWPHQWSHULRGDOODQLPDOs’RYDULHVZHUHDVVHVVHG IRUKLVWRSDWKRORJLFDODQGKLVWRFKHPLFDOLQYHVWLJDWLRQV$OVRDFWLYDWLRQRI12V\QWKDVH 126 LQWKHH[SHULPHQWVZDVVWXGLHGXVLQJ1$'3+GLDSKRUDVHWHFKQLTXH
5HVXOWV 7KHRYDULHVRIUDWVWUHDWHGZLWK/DUJLQLQHVKRZHGSRO\F\VWLFFKDUDFWHULVWLFVLQ contrast to those collected from control or naloxone pretreated groups, based on image anal\VLV$GLIIHUHQFHLQHQ]\PHDFWLYDWLRQZDVDOVRVKRZQLQWKHVHFWLRQVWKDWEHORQJHGWRWKH JURXSVWKDWUHFHLYHG/DUJLQLQHZKHQFRPSDUHGZLWKWKHSUHQDOR[RQHDQGFRQWUROJURXSV &RQFOXVLRQ %DVHGRQWKHVHUHVXOWVZHEHOLHYHWKDW12PD\SOD\DPDMRUUROHLQWKH SDWKRSK\VLRORJ\RI3&26 Keywords:3&261LWULFR[LGH/$UJLQLQH1DOR[RQH1$'3+'LDSKRUDVH Citation: Hassani F, Karami M, Jalali Nadoushan MR, Eftekhari Yazdi P. Nitric oxide-induced polycystic ovaries in the wistar rat. Int J Fertil Steril. 2012; 6(2): 111-116.
Introduction 3RO\F\VWLFRYDULDQV\QGURPH 3&26 LVLQWURGXFHG DVFRPPRQHQGRFULQHGLVRUGHUWKDWDIIHFWVXSWR RI ZRPHQ GXULQJ WKHLU UHSURGXFWLYH DJHV 7KLV disoreder is manifested by heterogeneous clinical feaWXUHV DVLWFKDUDFWHUL]HGE\KLUVXWLVPLUUHJXODU PHQVWUXDO F\FOHV DQG LQIHUWLOLW\ ,Q DQRYXODWRU\ ZRPHQVXIIHULQJIURP3&26WKHSURPLQHQWRYDULDQ VLJQVDUHOLVWHGDVWKHIROOLFXODUPDWXUDWLRQDUUHVW
Received: 14 Jun 2011 , Accepted: 5 Feb 2012 * Corresponding Address: P.O.Box: 18155/159, Deptartment of Biology, Faculty of Basic Sciences, Shahed University, Tehran, Iran Email: [email protected]
DQG LQVXOLQ UHVLVWDQFH 7KH ODWHU SKHQRPHQRQhas EHHQUHODWHGZLWKLPSDLUHGSURGXFWLRQUHOHDVHRI HQGRWKHOLXPGHULYHGQLWULFR[LGH 12 DQGLQFUHDVHG OHYHOVRIHQGRWKHOLQNQRZQDVPDLQPDUNHUVRIYDVFXODUGLVHDVH NO is indicated as an important paracrine mesVHQJHUWKDWSDUWLFLSDWHVLQVHYHUDOSK\VLRORJLFDODQG SDWKRSK\VLRORJLFDOHYHQWVLQWKHHQGRFULQHRUJDQV This molecule is a free-radical produced from the oxi-
Royan Institute International Journal of Fertility and Sterility Vol 6, No 2, Jul-Sep 2012, Pages: 111-116
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dation of terminal guanidino nitrogen of arginine by WKHDFWLRQRIQLWULFR[LGHV\QWKDVH HQ]\PH 126 7KHPROHFXOHLVZHOOGRFXPHQWHGDVDORFDO inflammatory generator and included with the factors introduced as those responsible for the RYXODWRU\ SURFHVVHV DQG WKH 3&2 V\QGURPH DV ZHOO 1DOR[RQHDQDUFRWLFGUXJLVXVHGDVD QRYHODQWLLQÀDPPDWRU\DJHQW 7KLV VWXG\ HYDOXDWHV WKH LQIOXHQFH RI /DUJLQLQH D SUHFXUVRU RI 12 LQ 3&26LQGXFWLRQ LQIHPDOH:LVWDUUDWV1$'3+GLDSKRUDVHSURYLGHV YDOXDEOH ELRFKHPLFDO GDWD UHSUHVHQWLQJ the participation of NO in this phenomenon. The marker is classified as the best indicator RI 126 HQ]\PH :H KDYH DOVR H[DPLQHG WKH metabolic status in L-arginine-exposed rats and HYDOXDWHGWKHHIIHFWRIQDOR[RQHLQ/DUJLQLQH treated rats.
Materials and Methods Animals In this experimental study, the animals were eight-week-old female Wistar rats (weight: J SXUFKDVHG IURP 3DVWHXU ,QVWLWXWH RI ,UDQ 7HKUDQ ,UDQ $QLPDOV ZHUH UHWDLQHG under standard conditions in accordance to the *XLGHIRUWKH&DUHDQG8VHRI/DERUDWRU\$QLPDOV $QLPDOV ZHUH PDLQWDLQHG LQ D VWDQGDUGWHPSHUDWXUH Û& DQGKRXUVOLJKW dark cycle with food and water ad libitum. All H[SHULPHQWVZHUHDSSURYHGE\WKHORFDO(WKLFDO &RPPLWWHHDW6KDKHG8QLYHUVLW\ Drugs /DUJLQLQH QLWUREOXH WHWUD]ROLXP 1%76 DQG ȕQLFRWLQDPLGH DGHQLQH GLQXFOHRWLGH SKRVSKDWH ȕ1$'3+ ZHUHSXUFKDVHGIURP6LJPD&KHPLFDO &R 6W /RXLV 02 86$ 1DOR[RQH K\GURFKORULGHZDVSURYLGHGE\WKH7ROLG'DUX&R7HKUDQ ,UDQ .HWDPLQH DQG [\OD]LQH ZHUH REWDLQHG IURP9HWHULQDU\2UJDQL]DWLRQ7HKUDQ,UDQ Papanicolaou (PAP) stain )HPDOH UDWV KDYH D GD\ F\FOH LQFOXGLQJ proestrous (12 hours follicular growth and peak esWURJHQ SKDVH HVWURXV KRXUV RYXODWLRQ SKDVH metestrous (12 hours corpus luteum secrets progesWHURQH SKDVH DQG GLHVWURXV KRXUV FRUSXV IJFS, Vol 6, No 2, Jul-Sep 2012
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OXWHXPUHJUHVVLRQSKDVH :LWKRXWH[SRVLQJZLWK male rats, the female rats are always in the diestrous phase; their phases will progress only DIWHU PDWLQJ ZLWK D PDOH UDW :LWK UHJDUGV WR WKH DERYHPHQWLRQHG H[SODQDWLRQ ZH FKRVH YLUJLQ UDWV LQ RXU VWXG\ %HFDXVH YDJLQDO changes may appear through the experiments, the animals’YDJLQDOVPHDUVZHUHDOVRH[DPLQHG during the entire experiment by the Papanicolaou 3$3 VWDLQ,QWKLVSURFHGXUHWKHVPHDUVREWDLQHG E\YDJLQDOZDVKLQJEHWZHHQ DPDQG pm were stained using a PAP stain for humans, DVGHYHORSHGLQSUHYLRXVVWXGLHV Three W\SHVRIFHOOVZHUHUHFRJQL]HGLQWKH3$3VWDLQHG smears: i. round and nucleated epithelial cells;ii. LUUHJXODUFRUQL¿HGFHOOV LLLOLWWOHURXQGFHOOV OHXNRF\WHV 7KH FHOO SURSRUWLRQ LQ WKH VPHDU ZDV used to determine the estrous cycle phase as preYLRXVO\EHHQSURSRVHG Drug administrations )HPDOH:LVWDUUDWV J DWHLJKWZHHNVRI DJHZHUHUDQGRPO\GLYLGHGLQWRWKH/DUJLQLQHGRVH JURXSV PJNJ Q LQ HDFK DQG D JURXS WKDW ZDV SUHLQMHFWHG ZLWK QDOR[RQH PJNJ Q SULRUWR/DUJLQLQHDGPLQLVWUDWLRQ7KHGUXJ /DUJLQLQHZDVLQMHFWHGLQWUDSHULWRQHDOO\ LS RQFH GDLO\GXULQJDSHULRGWKDWUDQJHGIURP WRGD\V 1DOR[RQHZDVLQMHFWHGRQFHSHUGD\PLQXWHVSULRU to L-arginine administration. The control group reFHLYHGVDOLQH PONJLS LQVWHDGRIWKHGUXJV Surgery procedure for removal of ovaries 7KHWUHDWPHQWJURXSVZHUHDQHVWKHWL]HGE\GLHWK\OHWKHULQDVSHFLDOGHYLFH7KHQDFPPLGOLQH incision in the lower abdomen area was performed. 7KHRYDULHVZHUHFDUHIXOO\H[DPLQHGELRPHWULFDOO\DQGGLVVHFWHGRXWWKHQFROOHFWHGLQD¿[DWLYHIRU further histological and biochemical analysis. +LVWRORJLFDODQDO\VLV )RUKLVWRORJLFDOLQYHVWLJDWLRQVWKHRYDULHVZHUH ¿[HG LQ D IRUPDOLQ VROXWLRQ DQG SURFHVVHG ZLWK D WLVVXH SURFHVVRU WKURXJK SDUDI¿Q HPEHGGLQJ6HULDOVHFWLRQV P ZHUHSUHSDUHGZLWK a rotary microtome. The slides were then stained XVLQJ WKH KHPDWR[\OLQ DQG HRVLQ + ( VWDLQLQJ PHWKRG DQGFOHDUHGZLWK[\OHQH$IWHUPRXQWLQJWKHVOLGHVZHUHHYDOXDWHGZLWKDOLJKWYLGHR SKRWRPLFURVFRSH 2O\PSXV 86$ DQG D YLGHR
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SKRWRELQRFXODUDWWKHGHVLUHGPDJQL¿FDWLRQ +RUPRQHDVVD\V Blood sampling for serum parameters At the end of the experiment, the rats were anesWKHWL]HG E\ LS LQMHFWLRQV RI PJNJ NHWDPLQH K\GURFKORULGH DQG PJNJ [\OD]LQH +HDUW EORRG VDPSOHVZHUHREWDLQHGEHWZHHQ DPDIWHU DQRYHUQLJKWIDVWWRDVVHVVVXJDUOLSLGDQGKRUPRQDO SUR¿OHV$OOVDPSOHVZHUHNHSWDWURRPWHPSHUDWXUH IRUDWOHDVWPLQXWHVWRIDFLOLWDWHWKHFORWWLQJRIWKH VDPSOHV7KHVDPSOHVZHUHWKHQFHQWULIXJHGDW JIRUPLQXWHV7KHVHUDZHUHVWRUHGDWíÛ& IRU IXWXUH DVVHVVPHQWV 7KH OHYHO RI VHUD DJHQWV >KLJKGHQVLW\ OLSRSURWHLQ +'/ ORZGHQVLW\ OLSRSURWHLQ /'/ JOXFRVH FKROHVWHURO DQG WULJO\FHUides] were measured based on competition binding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Û& IROORZHG E\ FU\RSURWHFWLRQLQVXFURVH7KHQVSHFLPHQVZHUHVHFWLRQHG ZLWK FU\RVWDW PLFURWRPH DW Û& P IUR]HQ VHFWLRQV DQGPRXQWHGRQSRO\/O\VLQHFRDWHGVOLGHV 7KHVOLGHVZHUHULQVHGZLWKEXIIHUDQGWKHQ stained using the nicotinamide adenine dinucleotide SKRVSKDWH 1$'3+ GLDSKRUDVH WHFKQLTXH WR YLVXDOL]H126DFWLYLW\%ULHÀ\WKHSUHSDUHGVOLGHVZHUH LQFXEDWHGZLWKVKDNLQJVWDWHLQD7ULWRQ; LQSKRVSKDWHEXIIHUIRUWRPLQXWH V 7KHVWDLQing was then performed by incubating the slides in a solution containing equal parts of nitro-blue tetra]ROLXP 1%7PJPOLQEXIIHU DQG1$'3+ PJPOLQEXIIHU IRUDERXWKRXUVDWÛ&8SRQ UHGXFWLRQE\1$'3+GLDSKRUDVH1%7\LHOGVDEOXH IRUPD]DQWKDWLVYLVLEOHE\OLJKWPLFURVFRS\&RQWURO specimens were assessed using the same procedure except that the specimens were placed in an incubaWLRQEDWKGHYRLGRIWKHPDUNHU1RUHDFWLRQWR1$'3+ZDVREVHUYHGLQWKHFRQWUROVDPSOHV7KHWLVVXH samples were then dehydrated in ascending series of
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Results Papanicolaou (PAP) stain 7KHHVWURXVF\FOHSKDVHRIIHPDOHYLUJLQUDWVZDV determined as diestrous after using the PAP stain )LJ 5RXQGQXFOHDWHG HSLWKHOLDO FHOOVZHUHWKH PRVWDEXQGDQWFHOOW\SHREVHUYHGLQWKHVPHDUV.
Fig 1: Result of Papanicolaou (PAP) stain of female virgin rat vagiQDOVPHDUVLQWKHGLHVWURXVSKDVH7KHDEXQGDQFHRIOHXNRF\WHVLQ WKHVPHDUVDPSOHVLQGLFDWHWKDWWKHF\FOHSKDVHLVGLHVWURXV
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+LVWRORJ\ The rats’ RYDULHV IURP WKH /DUJLQLQH GRVH JURXSV PJNJ H[KLELWHG VPDOO IROOLFOHVLQWKHHDUO\GHYHORSPHQWSKDVHLQDGGLWLRQ WR WKH IROOLFOHV WKDW VKRZHG HYLGHQFH RI HLWKHU atresia, large cysts with thickened granulosa cell layer, or large cystic follicles with scant JUDQXORVDFHOOV )LJ% 7KHVDPSOHVRIWKRVH SUHDGPLQLVWHUHGZLWKQDOR[RQH PJNJ )LJ & VKRZHGDUHGXFWLRQLQWKHLQFLGHQFHRIF\VW formation. The control group exhibited a follicular appearance depending on the phase of the cycle; in diestrous, only secondary follicles and fresh corpora lutea were seen. Atretic folOLFOHV ZHUH DOVR REVHUYHG LQ WKH SKDVHV RI WKH control group. The corpus luteum was absent in DOOFDVHV )LJ$
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Fig 2: Pictures of ovaries from control (a, A), solely L-arginineWUHDWHG % DQGQDOR[RQHSUHWUHDWHG & UDWV%DUVEHVLGHWKH VDPSOHVVKRZPPYDOXHV7KH¿JXUHVDUHVKRZQXQGHUPDJQL¿FDWLRQ>D îREMHFWLYHRIDQ LQYHUWHG SKRWRPLFURVFRSH $ %& 2O\PSXVSKRWRPLFURVFRSHDWî@
1$'3+GLDSKRUDVHUHDFWLYLW\ 3RVLWLYH 1$'3+GLDSKRUDVH UHDFWLYLW\ ZDV REVHUYHGLQWKHVWURPDDQGLQWKHWKHFDFHOOOD\HULQWKH RYDULHVRI/DUJLQLQHWUHDWHGUDWV )LJ$ 7KHUHDFWLYLW\ZDVDOVRREVHUYHGLQWKHPHPEUDQHJUDQXORVD FHOODUHDRIWKHIROOLFOHVLQWKHVDPSOHV,QOXWHLQL]HG RYDULHV ZHDNHU GLDSKRUDVH UHDFWLYLW\ ZDV REVHUYHG in patches at the periphery of the corpora lutea. The UHVSRQVHWR1$'3+GLDSKRUDVHVKRZHGDWWHQXDWLRQ LQQDOR[RQHSUHLQMHFWHGVDPSOHV )LJ% ,QDFRPSDULVRQDQDO\VLVFRPSOHWHGE\,PDJHWRROQRVLJQL¿FDQW UHDFWLRQ ZDV REVHUYHG LQ WKH FRQWURO VSHFLPHQ )LJ& ,WVKRXOGEHQRWHGWKDWWKHDUHDVRILQWHUHVW FRQWDLQHG 12JHQHUDWLQJ HQ]\PH DFWLYLW\ DQG WKDW WKHDFWLYLW\RI126ZDVLQDJUHHPHQWZLWKWKHWLPHRI rupture and cystic formation.
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12LVFDWHJRUL]HGDVDQLPSRUWDQWLQWUDRYDULDQPHGLDWRU WKDW DIIHFWV WKH RYXODWRU\ SURFHVV DQG regulates the functions of the corpus luteum. The present study has lent further support for a functional role RI12LQWKHRYDULDQIXQFWLRQV$FWLYDWLRQRIWKH126 HQ]\PHZDVGHPRQVWUDWHGXVLQJ1$'3+GLDSKRUDVH 7KHSUHVHQW¿QGLQJVVKRZHGWKHDFWLYLW\RIWKLVHQ]\PH ERWKLQWKHVWURPDODQGWKHFDOOD\HUVLQRYDULHVWUHDWHG with L-arginine. 7KLV VWXG\ SURYLGHG PRUH HYLGHQFH IRU IROOLFXODU atresia, as well as the production of large cysts due to 12WUHDWPHQWLQ:LVWDUUDWVZKLFKDJUHHGZLWKDSUHYLRXV¿QGLQJ'HVSLWHWKHYDVFXODUHIIHFWRI12ZKLFK IRUPVWKHKLJKO\UHDFWLYHF\WRWR[LFSHUR[\QLWULWH LW PD\PHGLDWHWKHFHOOXODUF\WRWR[LFLW\RIRYDULHVE\WKH 3&26SKHQRW\SHHIIHFWDQGYLDF\WRWR[LFSHUR[\QLWULWH NO production and formation of the free radicals may KDYH VRPH LPSRUWDQFH LQ WKH RYXODWRU\ SURFHVV 7KLV IDFWPD\IDFLOLWDWHWKHFDWLVVXHHYHQWV7KHWXQLFDDOEXJinea of the follicle wall may be one of the symptom’s VLJQVDVEHHQQRWHGSUHYLRXVO\ ,QWKHSUHVHQWVWXG\ WKHVHDUHDVKDYHEHHQIRXQGWRFRQWDLQ12JHQHUDWLQJ HQ]\PHDFWLYLW\DQGWKH126DFWLYLW\DJUHHGZLWKWKH time of rupture and cystic formation. ([FHSWIRUWKHOHYHORIVHUXP/'/ )LJ QRVLJQL¿FDQW FKDQJH LQ RWKHU OHYHOV VXFK DV JOXFRVH RU EORRG VXJDUZDVIRXQG1DFXOHWDO VKRZHGDQHJDWLYH correlation between NO and markers of glucose hoPHRVWDVLVLQ3&26SDWLHQWV7KH12OHYHOVDSSHDUHGWR
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be regulated by endogenous estrogens because a higher OHYHOZDVGHVFULEHGLQWKHIROOLFXODUSKDVHWKDQLQWKH OXWHDOSKDVHRIWKHPHQVWUXDOF\FOH 7KHVHGDWDSRVVLEO\ VXJJHVW WKDW WKH HVWURJHQ OHYHOV LQ WKH GLHVWURXV phase may interfere with NO secretion, which is preVXPDEO\UHODWHGWRWKHSUHVHQFHRI3&26 7KRXJKQRVLJQL¿FDQWFKDQJHLQVHUDOHYHOVZDVIRXQG the administration of naloxone prior to L-arginine led to D GHFUHDVH LQ WKH OHYHO RI VHUXP /'/ )LJ DQG DWWHQXDWHG12SURGXFLQJDFWLYLW\ )LJ 7KHVH¿QGLQJV possibly demonstrate that the NO molecule, as a proLQÀDPPDWRU\HOHPHQWPD\LQGXFH3&26E\DFWLYDWLQJ LQÀDPPDWRU\IDFWRUVLQRYDULHVDQGWKHDQWLLQÀDPPDtory agent naloxone affects the endocrine and metabolic PHDVXUHVUHOHYDQWWR3&261DOWUH[RQHDGPLQLVWUDWLRQ has been shown to result in reduced food intake in anGURLG REHVLW\ 0HWDEROLF DEQRUPDOLWLHV VXFK DV hyperinsulinemia, insulin-resistance, and obesity, are OLVWHG DV FRPPRQ IHDWXUHV RI 3&26$ OLQN EHWZHHQ RSLRLGVDQG3&26UHODWHGLQVXOLQUHVSRQVHWRJOXFRVH ORDGKDVDOVREHHQSUHYLRXVO\VXJJHVWHG
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