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Effect of Metepa on the Reproductive Potential of Rattus norvegicus
Dissertation submitted in partial fulfilment for the degree of
Master of Philosophy tn
ZOOLOGY of
The Aligarh Muslim University, Aligarh
By
MAHIDHARA NIRMALA
DEPARTMENT OF ZOOLOGY
Aligarh Muslim University, Aligarh September 1976
;ssi: «** UNIWWi'^' '
DS29
Bfsscf Of m^mx OH ®E sspHJroofnm ^o^smAh of
Stt1»ai"ll!ea in |>arllal fttlftliaQ»t tOT His Itsgsje® of
Ma0l!9» of i^hSXami^y
in
Zoology
of
Bs pajptaeat of Zoology
t eerttfy liiat •^Iftet of Me-fcepa <m ^ e 3jepro6»otl've
potsattal of Batta^ norve^ffla" i s 1fae original wik of
IE as liahiShaza Hizimla and I s ^Itai^aLo for ttie parHaX fulfil
ment of iJiQ aagreo of Master of Hiilo^pihy i» Zoology of
•die Allgaifti Httslia tfalimrsltyt Aliga^. "Sie wojfc has toeen
do&e under my supervision*
^mm VU AXBAMt ph#l)*(HotPe Saoe)
Readsr Dopartneat of Zoologyi i a i ^ a * IMsllm tJai'rorslty Allga]^*
I am deeply tnatelitBa to Dr« XJmm M# A<3haEdt ^ o has
gupervisdd 1^8 w03 « Hie imliialile dtagga8tl<m8t cri t ical
advloQ and ccmetaat encou3?ag©aettt w33Pa of gxeat iaportane©
for itie ooiapletLon of 1hl0 vosk*
1 am gmtefua to Pxof, S«M* ilaia^ Head of tio Depaziiinentt
for providing neooaoary lafeojjatory faoilitlesf
My slnoex^ ISsas^e to Mr« tEaxit Haa^ and Hlsa StiamgL
Hi Aaaarl for Ifeeir cooperatlan during Ih© course of pre^mt
stiidy« 1 also iStimik Miss S lxt ifaleee and U s s Zassaln Hanii
for liieir help ia "tt e preparaMon. of 1A,SOLIS0Q^
Hnaaoial aid for this w03 ha© heea proidd©d hy ^©
Iftii'verslty Grants C^aaaissian in Ui© lOxm of a Junior Begaarcai
fellow^ip,
X am gratsfkO. to Uw» Qasii Mohd, Shasclm for hi© attentiire
typing of Ihia dissertsticas.*
poir,,!gB,^ %§ fage
Iiiat of IJatiiles and iisar^s ••• ( i l )
T!al3le of Contea-fes
!• latwauottoa and Heview of Mteiatare 1
11 • MateilQlo aad ae-mode •»» 11
i n . Obseivatioas and Ha^at© •«• 15
!?• M^cuggloai ••« *•« 20
% Conciltistoaa #•• •»• 30
'Vl. Susjaary ••• • • • 51
'?I1, Beffesences •*• ••• 53
MtlMmmMIINI*
^il)
X, Bffeet of Itetepa on Hi© j?epa?oftietlve
(CheiaieaX adSiils '^TOaoi^l^ t© males mXf}*
XI # Effset of Ifetspa oa -8ie i?ep»oa»etlire polmttal of M^MM msSMMa* (Cheiaieal a&ainist0i?ed oaiaXly to feiiales <mXy)
III* Bffeot of M#t®pa im Itie Isody «el#^t of
X# Ifj^et of astopa cai 1iie repieoAttottve pol^alial «f Batttts m3!^fim^ i tm&immt to males),
XI* Bistogxam ^o^dng %# ef:0aot of mo'^pa om l^e m^ToGsLQiim potential of j ^ t ^ s nogroglcas
( t p t a t a a t to iaal#s)»
XIX, Effeot of metepa caa iti© impsoaactLve po'^i t lal o^ lat taa noyvi^c^,^ (faasstamt to ffeiaalea)*
I?# Histog3?^ sho^dag llio tffeot of metepa oa tb® TOprodtaotlire potential of .B^tt^f neg^g^mQ. (Ififeataeat to feu^es).
rmyttX* Histology of ife© t totss .
IHTROOTOHQH AHD HBVIEW Of LIffiBAWlS
met® i s a VQvy great need of deTOlopiag soltaTsOLe
m0a»s for aiPtlfldally segulatlns Ihs lolrtii sat© of tioee
laiia vertolwal»g Uiat l l w In soainatajKillstlc aitaaHoas
and hava 136 come troulalosoma to aaa« !Ehia ^ u l d lie a
dQ0t3?aia.e piocadujfs for equallsslng sosto of nian*8 oaiifLiotliig
Ijateiactlona wl-fti -tha ecologioal Mots and i t ^ u l d help to
aaaiage a haalijiy environmejit la perpetttity.
Bodeats axe caie of laan's Jiiost important eoazzdea*
'Siey destroy a sisaaKLe part of hi a growing food onopSf
attack stored food and other prodacts and toTment -^Q
liinestock by spreading diseases* I t i s imlaentt 12iereforet
•mat liielr ooatrol i^ould be brou^t about*
Bodent ooatrol has beooae a oomplax technology now
and oOTipriQes diiefly of liie use of pestLdLdasp repellants
and oheaosterilants* All -ftiese are agents of biological
control tAdcfc in -ftie long run prove more efficadous*
tJpto now man has just wanted to destroy quicfcly, bat now
he sometiiaes wants to ^eep Ifeem allWr so Ifeat may oaa aid
in -^eir ova dastcuotloa, Sudi self*-d©st3!uetiQa imy be
• 2 -
aeoompU^ed lay sexually steillialng "bsit not killing Itie
pest ficpedes %il^ certain kinds of cheialeals. Ch ialcalQ
ynidti haim tfeiia unique property axe often ayeferased to ai
CHKIOsfSaiLAHfS* More over Tjy using a eomliinatitm of
sl^xilants and conTOntlonal conteol meUiodet i t may Ije
poe^tflLe to obtain sj^eigistlo effects* vbere -Sie degree
of control adiieired will greatly exceed tfcie m& of ihe
independent effects of each jseliiod alone*
Ihe ^verte^EBtes liiat we daestfy as pests have very
steep population groirei curves* Populations of laany of Itie
vertebrate pest species are probably a t or near aie levelled
off top of ^ e classical growlh curve, !S?o ar t i f ic ia l ly pu^
-Qiem off Ihe plateaUf by biogenetlo control or any o-e^er laeansf
on to "^e precipitous slope of ihe sigaoid grow^ curve i s
of no avail unless the biogenetic control effort be unrelenting
or else i t be accompanied by a ocmourrent control of Itoe bio t ic
requirements Ihat nurture Ihe grow^ curve and eurvlval rate*
A half sterilized bacterial cul"t»re does not remain sterile for
longt nei-Qier does a reproductlvely Inhibited populat4<m of
dua^ fed rata (We'ftierbeet 1964),
BSany scientists have postulated Ihat -fee presence of
a given number of sterile individuals in a population exerts
•• 5 **
a audi sapeat$r tetologioal control -j^vesmT^ oa Itiat poptalattoait
ifaan If liie same aualser of f&rtll© Individuals WBXB »mowd»
'Scila cojxoept Ifeat 1iie staill© inai-widuals wtii<& f«@iaalat not
oaly f ^ l to oontnimte to l^e next gensratlon l)at meaav^le
also compete for gepacet food aad eodol OT&&T I S c»>a?3?oct
Mologloally* al&oe I t i s laased m popiilatton pzlnoiplss of
density depeadesace and -Sis edgaoid groy^ rnvTe*
!Sie credit for introdaoing sterile individuals into a
natural populaticai to obtain control of 1fae spooles goes to
Koipling who in t937 proposed his "sterile male tedmitus"
and applied i t to -fee screwwosm fl ies (Snipling» 1960)*
According to him, •insects Ifaat are sterilised cannot
reprodiice and -axus such effect i s e<|uiiraleat to kill ing -the
organiaas- In adaitiaa» -fiie sterile insects compete wi1h
ihe noxiaal insects to furitier decrease -^e (dances :^r
reproduction, Ihis honus effect repxeeents Itie greatest
adOToatage over tbe coaventicaaal i^y of controlling insect
populations* In addition, howeverp sterile Insects in Ihe
population are capaiae of limiting reproduction hecause of
• tlme and space** effectst factors iMdh are absent in the
killiaag procedure "• Hho geuae principle can he , applied to
rodent8«
4» 4 •*
%e appllcatlim of niaaljBra gaa as sea i3?»ltaat and imsctoaat
to liie f i r s t irfQxld war. Mi-ii tie adireat of Ih© second xOLd
w« and lacauslon of K aiistard aad aaaaogaes afflwmg pol^mtlal
m r gas©St a ca3?9ful i?8»appa?aisal of -Si© ejsteuic toxiisllsy of
^ 0 gtoVLp m& imdertaken*
diofflos-fesrilsnts laay "b© dsftaed as dienjloal oompouads
•vfoldi psasic© or ^ i t t r s iy ©liaiiial!© -^e i^produoHire oapactty
of Ifc© aaiaal to ts^ldi Itiey ar© adotnlgtered* ^©y a^y
eff©et caaly o»e sex of a ©©aaially aseprodaclag ^almal or 1)oifa
Q©3Qi©, Hitir aff^ot may l>e tsapoi'scey or panBanoati iiaaeaiate
or delaysd, ®i© tens cbemostexHaat doaa not liidlud© chamlcaX©
^Icfe «o«id# diree-ay GT indirtotilyt latarfer© wllto oatliig,
Oa Hi© olfe©r haad i t aay 4a<a.ad© iaat©rlsas ifcl*^ do not interact
direemy witi geas oell© or gtmada*
Oisiaical© ha-^ing s teniant a c t l ^ l ^ vary tddoly la
stmoturet Isit oaa ^ grouped la to fl-s^ ^galfloaat oategorle»-
al^;^atlJig ageatat aatl]iietat70lit©s« radiomlia&llo oosipouadst
ffll^-a.c polsms aad j^acsllaaaoua agaats# Metspa* tspat
IMotepa aad apholate are ^Ik^^tteg ageats* Alk^a'tlQa
Implies Uie latrodaotloQ of a hydrocarbon r a s c a l o f t ^
ooataiaiag eleaeata oHier Itoaa carlacm aad hydrogea i^to a
3 *•
molecule under phyeiologio coadttl<ms» Compounds capaMle of
aocoapli^lag mtSi jwaotlons axe aUcj^atlag agents, Ihe
most Important and most numerous group of alkylating agents
are liis derivatives of "Aziridiae'' (eltisfleneiBiiaes)*
According to "ftie availaWLlity of Ihe free electron pair on
"the rinu H , aeirldlnes can "be divided into two groups*
U Basic and 2« Heutral, Most effective eSiemosterilaats
Iselong to Hie neutral category (LaBrec(|aei 19^1) •
Alkylating agaits are notaTSle for •ttieir delayed effect accompanied by a selective action a^ ins t some proliferating
tlapneas haematopoietic cells of ihe bone narrow and lymphoid
tissueSf 'tie intestinal mucosat gexm cellst embryos and
tumd^s (Hayesf 1964)» In eapenmontal aniaalst l ike , mouse
((^ttanach, 1957)1 rabbit (Pox* £ i ^ * t 1963) and dog (Hendryi
aJUik-i 1951) an entire range of sterlliadng effects has been
produced by ^lk3Q.atlng agents.
« 6 -
yiapp ^ A» <1962) have slaiaied -ane as-talxjllsa of
meteps • |^^ i» house Hie© aad in laarvae anfi adults of Ihe
iao^wltO0a ,.»,§.s ,1fiym?. fl QsamMM* ^®sa Iniragtlg^tora
found 19mt ^& Giiomloal itias fspldl^ absorbed la hou^fl ies
following topical afiadidetiBtlosi and jsipldly detojdfled
followiJ3g a1)sozp1io», AlJout 50SS TI®0 degsadad wlliiia tvs)
hours, iBjeotad fl ies dagmdsd ttie cheadoal a t an e-roa moif©
lapid »ato* Degradation M&Q moj© ihan S3i complete in
24 hours* Bolh dttsceptiTa.© strains and organopho^hate
r&sistant straina mve uasd* Ttie ratos of dagradation were
siadlar in Iho aasceptiiae fly strain and tn lixe organophoo-
phats resistant strains* IJiey also found tJmt midb. of -tti©
axorsted xadio aotiTO mataxial appeared to lie unmataholised
aetepat althou^ en unknomi compound end inors^Laic pho^ihatea
•ware also detected. Of the material exoreted^ ©ore iiian 90^ 32 of -fee total £ recovered in 24 hours 'ma eliminated ^i-thin
e i ^ t hours of treatasnt.
la -Qie mosquito larvae alsot "^o eiieiaical started
degrading rapidly and ii«as almost ooiarpletely degraded vdlhin
48 hours (^lapp sSi Sk*» 1962) • Hovie ver, loea^irahle amounts of
radioaottvity remained in ttie. insects -^irou^out Ihe pupal
and adult stages* Ihe adult mosquitoes also degraded tbie
mm f tm
diensical sapiaO-y bi t unicfa of Urn label xeiaalaed la -^m
tasBots. S rom Ihese Qjgperlmsiits i t appears "Siat iMtepa i s
degjna^d by both laosquitoes oafi boueeflies aad Itmt oae
of "tiie d^graaatLon proctaotsf possibly PO t i s relaooarpojrataa
iato 1ii0 aonaal mstabolilss.
In Uiis cBiae study Uie mslabolisa of me-tepa ia laioQ
w,e also iaTreetigated (Plapp £ l Ji^tt 1962)* 'BXQ mmltQ ^oved
Itmt 141© labelled cheiaieal was ©xcsee- fi almost as rapidly la 32
sriloe as la houseflles. Mogt of Hi© P ms eacoxeted la liio
uriae aad %ias i^llectad wltii Iha twelve hours aftsj? trea1s»atf
Qoly a mall aaouat was xecowred ia Hie flaeces, l a Ihe
exoretory p3*odttctSi waaltered metepa aad oae saa oy breakdowa
product, prsfiwaably iaojcgaaio phosphate, were detected by
paper daromatogmphy,
la houeefXies oetepa a t 1^ was fouad to aubstaatlally
sfeortea 1he l i fe spaa of liie ias^cts (Murvo^i LaBreocjue aad
aailtof 1964) • At 2,5j5 aad 55 i t was seea to oscam e te i i l l ty
ttoea the treatment was done oa pupal stages (GoudCf 1964) #
Studies on comparistm of quaatltatlve effects of tepa* aph^a^te
aad metepa on oale houseflies ^owed i^at laetepa was -^e least
effsective of -fee "eizee cfcendoals ia steriliBiag the iaseots
(Ghang aad Boxfeoroo, 1964) •
!3ie sterilisdag aott-vity of metapa ia buffea?ed solutioas
does aot al ter significantly wi-ti cliaag© of pH (Bojko^Ct Chang
fABmB 1
Iffeot of M©i50pa m Ihe Iteproauctlve Potential of Battai? mmea^mu
(QieialoaX adiainist6a?©a oaslly to aales coaly) <iMtw«Wi»iiiiii%*i»wiwi>««i<iip<iii«Miw»«^^ ' mmim»mm^u»»'»»m!i*»*»m>Mmmiimmmmmmimiii\ IMHUWIIH
iro« MT» MBI A M^ Of offapriiig Aea Ho« of offgpiiag Oim dilu^ <E^100 ga l»,wt.) ailutea with iiieOHOIi* ted wtlfe msmiLBD WAfSa*
t 9
6 8
5 8
4 7
HHi 6
Hill ta
B1I» lipi
t*
2,
3*
4»
5.
O f
?•
8.
(0C3ii!CHGI.)
o^oa
0*025
0»05
0»10
0^20
0 * 3
0.50
• Bata TsasQd oa 'fiiajee replicate a of oacii*
«• 8 «•
and Iiimtoai!g# 1964) # la partially degmded solatloas tie
©tarlllsdug activity haa "been fotmd to tj© pjroporfcional to
-ii© coal^nt of intact mete|>a coatalned 1» Ihe soltitLan
<BoiScoi?«Ot Chang aad Limtottxsgt 1964) •
Iteiae and Sdwaldt (1964) conducted a oompi^ffla^ve 32
slaidy of Itie uptake and aetaljolisEt of laetepa - ? in 'fee
houaefLies and in two apaoies of moEKiuitoaa, Hie Xa^lled
compound m& rapidly absorlaed ftfoa the ^ a a s auiff&oe liy bo-gi
BipQciee of mo®iultO0s aa well ao Ijy liie houaefXiea* Ul8trit»-
tion of Ifa© laljelled ©etepa was sapid and general in these
ingDcta. IhB label waa found to Ije psjoportlonataly dietritjuted
according to falatl"^ wel^ta of ifee various l»dy secilona
which were investigated*
An extensive study of Ifae rate of absorptlont excretloa 52
and laetaboliam of laetepa^ has also l»8aa made by Chamberlain
and Hamilton (1964) in HXQ screwwoim fly and ihe stable fly*
I t waa found Ifcat six hours after eaq?osttre to -Sie etarllanti
•eie screw worn fly had absorbed oaaly ha l t as iau<4i labelled
laaterial in proporticaa to i t s sdae as 1tie stalle fly, explain^
lag iiie differenoes in Ibe dosage required to steril ize Use
two apeeiea#
- 9 -
Chromatographlo ezialyssi^ ( ChQml)0i*Xai& mi.& H$2slX-t(m«
1964) reviealed 1iiat liie principal aieta^llt^ ^ s i>ho^03:^e
acidt ]pBoa3L mairteir ocaataiBed aauy oilier ine1a'bolit09# At
i©ast four of iStm iinkaom aetalxaLites bad l»®i psegeat as 32
ooatanilnaiits la 1he puuiiaied aatapa f used ta IhQ slaidyt
Tait a l l ^Qm taiJaioiritte Incaeeased la qttaattty i» "fee fiecsal
imtoxial to levels high Him. liioae fouad ia l^e oxiglaal
aamplaa* I t has beea guggeeted (Plapp ^ ^ . » 19^2) Ihat «sa©
product may to© either the di- or mono a d d derliPatliTS of
laatdpa*
^3dc dosage of metepa to ra ts has t)e6a fotmd to t)e
136 mgAiS of hody ii©ight hy oral route aad 183 nigAs of body
v e i ^ t hy dermal roato (Gaines ead Kimborough, 1964) • Ihs
orgaa pnaar i ly affected by repeated snail doaes of lastepa i s
teste81 cmries aad bone marrow being damaged a t only higher
dosages (Grainss ead Kiaboreu^i 1964)*
Ifudi of Ihe studies conoerned wl-Si Hie effect of
alfe:3?latlzig agents m fe r t i l i ty have so tax been on the
administration of continuous doses of chwaieals to -^e es^erl*-
mental animals (<^Paig ^ jl^,, 1958f Gaines aad Kimborou^p 1964) #
•Bie present study \iaa undertaken to note -Qie effect of a
s in^e low dose of liie oheaosterilant metepa on 1fae reproductive
potential of nozmal male ^id female albino rats* &e problem
« to
has t»9a tadclod from laoih jfeproauotltB as tieXl as histologic
points of viBv* Uhls paeooe<Ju3re» has aa adimatago 0"TO3? "Sie
oQutlmioms a<3i!}iiiiatmtiosa of Ihe tdiosiioal as i t oaa ^ saocaa»*
ftaiy applied to lasge acale field atttdies iili0» I t \«?pXQ. "im
dif£l{»dLt and xa'tter ii^xaotloal to adidiilster ciiaiidoaXa
eostiimoudLy* Ihe woiSc i s ©apeoted to giTS an Inst^i t into
Ihe effeot of chooioals tm hvama Iseings and^ in ^m Img xWf
ajay h© applied to fe r t i l i ty control progassEaaea*
Ihs present study Indndes tie effects of ^Hi metepa
and 0-8151 alcohol on Itis raprodaotlirs potential of alMno rats»
- 11 -
5to determine Itie effect of metepa Of1V5003»"b¥) cm
tie apep3«>asietliire potential of Balaia norve^otts <alMiio ia t ) t
Ifee folloidn,^ sets of e3E|>8rim©nts wei« perfoiaed, SL»oe
metepa i s soluble Isolti ia water aa well as in alcohol, two
aets of e^r imente yem condtiotedQ In me, Ihe ^ t s vere
tpeated wt-Hi laetapa diluted ia dlsHlled «ater, \liile In ihe
oHiert '^ey mvQ gl' en metepa diluted in absolute ©Ihyl
alcohol (C^50H)» Biis vas done in ©ydea? to assess Ifee
effeet of alcohol I tself on -fee :eepi?odueti"9ie potential.
^3? -Sie experlaentsi l ^ l y gro^rif reproduotively
laatuxe, adult albino rata of laboasatory bred stock wa»e u^d«
Seven different concentsaHcaas of metepa i»e» 0»02 mg*
0*025 m$ 0.05 fflgf 0.10 mg$ 0.20 uig, 0*25 mg and 0*50 mg
(per too s»ams of body weight) we3?e gi-vmi.
Metepa slxxjfc las a liquid of Itie con^ntxaticm of
1 graa/t a i l l i l i t r e . By -thousand fold dilutlont i t ms
brought dowi to ariLlligiam l e ^ l and as auch as reguixed was
pipetted out by uderopipettes. 5hese doses were made in
double distilled water (a l l ^ass) and 100^ e'feyl alcohol,
dissolved in 40 lal of boiled lailk and orally fed to tie
aniiaals. Bie drug can be administered elUier ©rally or by
- 12
InjecUon. fhe oral meltiod was pseferredf sAneet i f large
aoal© Held woaSc i s imdertakenf ojsal feeding would loe more
practLoal*
I t was ascertained by t r ia l aad error laelhod •fimt
40 ral las *&© maadliaua amotiat ISmt a ra t could take in a t
a time* SOf al l tbie doses of aetepa imve dlasol^ed in
40 ssl of tjoiled milk. Milk %ias tjoiled in order to avoid
infeo"Q.on of any kind* Only one single do^ V&B giipsn after
\^i<!h nonaal feeding of food pellets aad gram was resumed*
5he anisals were "Siea alloyed to sate wi'ii untreated aniaals
of Uie opposite seat* Prior to starting lfee experiments, tie
nortaal reproductlire potential of alMno ra ts was determined,
for -tiisf on© of Ifce sexes was ^r&a 40 ml of plain milk
after ^idb. normal feediing was reguiaed* ISie eniiaal was ttien
allowed to mats.
In case Of eaq^erimental aeriesi wben laales were liie
sex tpeatedf they were allowed to sate wiUi a noraea female
in estrus. When the eacperiiaents were carried out on "tJie
feaalest Hielr estrus cycles were regularly diec&ed and ttiey
were allowed to mate during satru©. Hie estrus cyde lias
four stag&s *• diestrusp proestjsisi es-fapus and metestrus#
I t i s during es-taeus 'Qiat females of seasonal "tepeeders like
ra ts peiffilt oopulalAon. fhe^ stages mve identified by
13 -
aafeing a ^giaa l aoear tm a eleaa elitl©. Sie iaentlflcatlQitt
of different stages «aa ifecilltatea "by atainlag %e gaear
wliSti wtea? sol«M.a aidliaa TadaeC Aniline Blue 0.5 » osalio
acid 2.0 gas « Bie-tlllea mter tOO c©),
Ihe YagLnal ^ears were pyeparefi "by inserting a
toopper containing nonaal saline (0.9^ laca.) into -ftie enimal
and witbidsawing i t wltfe tie looigs* cells of -ene iragina*
Iheae cells were then placed on a slide and diied all {fitly,
^ e obseivation of -Qiese cells wis cai«led out after staining
- em in aniline blue for ten niinutes and airdrylng liie slide*
Vaginal mBB.v& %iere oHeoked for -fee pfessnce or alisence of
^erms a day after aaatlng,
%e l i t t e r slaSf i f bom, m.8 noted, Sie de^^elopment
ya.B followed end any external ataonaalitiea «®re looked for,
loT hi9la>logic studies the treated male ra ts vers
sacrificed forty days after Ifee trealaaent idUi metepa end
their testes fixed in Bouin*a fluid* fhe males were lef t
«i1^ Hie females for a m&mLmm period of forty days after
y^ioh -giey wore sacrificed even i f no offspring %mTo bom*
Hlocka were laade and sections of 5 u liiictoess were <a»t on
rotary miorotMie to s1»dy ^Q histologic ebangest i f any«
m ^ ^ oM
Slides were stained wi'fti Haeaatost^Htee-eostn meHiod as
gXvon laelows
Haematoxyjine (^r l iehe 'a) (Kaeiaa-^xylia 80 gaa t Alcohol
3^00 aa I Jotassltta alDua 3i 0 gas « Metllled ^jater 1200 afl. t
Glyceyol 1200 aO. i Slaelal acettc acid 120 ail)*
Haeiaatoxyliii was pxepared "by dissolving Uie elum in
water using heat and adding glyeeipol to I t ^liile s t i l l warm.
After coollngt haeaatoxsHne solution in alcohol was added
to i t in small volumee liiaking well Isetween eadi addition*
Lastly f a c i a l acetlo a<dd was added, Hie solution was "Sien
ripened In l ight f&r 6*8 weeks*
fhe motions were f i rs t terou^t down to water and
flmtlon artljfeets removed, llhe slides were "^en stained
in haeidatoacyline for five minutes and differentlaled in
water* Ifeey were -feen lef t in tap water t i l l colour developed,
®ie sections ^ r e Ifcen upgraded to 90^ alcohol, after yhtda.
Hiey were s'teined in alcoholic eostn for two atoutes# After
dehydrating Hiey were cleared in dove oil and mounted in
Euparol,
•* ^5 **
OBSSaTAKCms AKB HBSttLTS
a e llttser SIE© of Bajitag aoa^ideas I s 7 to 8 yoting
on©s per l i t t e r according to Piagalep KidfiJmaiaiir^y and
BamaslTma (1967) and 6 to It swing ones per l i t t e r according
to Nalljandoir (1970) • In al l i&c control experiments conducted
in -tols lal»ratory, -^e l i t t e r gtg® IMB a constant of 9 yoang
one©, IMs was» -eiereforet l^iiEa as %e nortaal reprodwotive
perfonaanc© of albino rate.
In ih& experlBJsntSf treatraaat of setepa on eittier
sex produced a detLnltSt aariced reduction in Ito© l i t t e r slae
as ^oya in itie graphs C^ss* I ^ I I I ) , In eaqperimental
eerie Sj toere laalos were treated, concantmUons of metepa
above 0*1 lag / tOO gm Ijody weigfctt produced no offgpring»
ttiough mating was observed to have taken place. In aniiaals
treated wllh lower concentratLonat i^ere was a graded and
unlfoaa reducttca in liie number of offspring per l i t t e r
as evident by Ihe histograms (Hga, l i t I ? ) . VH-Qi the
trealiasnt of ms'tepa laiasd in distilled water, "^e number
of offspring in a l i t t e r came down to 8, 8t 7 and 6 a t
concentrations of 0.02 si^t 0*Q^ agt 0#05 mg and 0,1 lag
per 100 gms of body weight respectively. Ihe effect was
even more pronounced Oien laetepa was adalnlste»sd in alcohol.
en 00
2
3ZIS aa i i i i
ttg« H M§^WM, ^oviag fi3 offset of ostepa Hid
a?
in
g t » t U W t t « l l < « I I I I I B , l *»T1
i • I I . .11 I ^ . . J I L •
M » i r i r « i r a i j ( j i i t K i ( a « k « k « 4 « y j r « . ' J » » « l , > l l , l ( « M « « H l i « » « » < » « J t l l « »
Ii':V:'.!'.'.-I.'.v!'..'.':.\S-.':-.v.V-
E l l » l ' a * « « « i i ' i a « « f i > i i « a « i l « s i i i k i p « « » l r » > « » « l i « » « « » » » » « » l l « » » l
Mi>ti J .v. i ' . ' f ! ' . ' V " ' . ' . ' t ' . ' f ' ' ' ' ' . ' . ' '
o at » t<^ vb
B
o «f
in o
in o < . o o •-
< CL °. •-O
O
o*
m ^ r» M ^
3Zisa3iin
• to "•
la Ifelg cms© -ai© »iMl3er of ottering in a ll-tter tms
i»^oed to 6t 5 mid 4 a t Ifee i^rat 1fer@© coaoeiitrattcms
3?e0p@etlv0ly \lille no young one© mre feom %hen "Qie
concentratloa ms mlsefi to 0,1 ®g laetspa per 100 gms
of bo5y w0l#t (febjL© I ) .
l ie yoiaig ones IKJXSI ii03?e a l l appaimntly n033aal»
ITo extemal atooijBalitles «ea?8 ofesenreS, Howe-ver it 'was
noticed "tiist 1iie progeny of tlie treated anin^ls vQm
definitely of dialler S1E©# 1h®i« m.B a reduotioa in liie
Ijody wei^t of Ibes© :?- indi-vidiials (fable I I I ) t bat i t was
not si^fieffiiiaiy laiqge. ^ e deirelopa^at iiss also observed
to be Bk&v&T than -ftie Qffs^ring of -Sie control aeries, U&st
noticeable difference wae in Ifae dewlopaeat of hair o per
•fee ^ i n of -^e ymmg ones* In "ttie eon1a?ola# UB progeny
started developing hair by -fee end of f i r s t «eek» In liie
ejsperifflsatal eeries» -iie progeny iiiim. alive) did not
develop any hair t i l l after ten day®, Sie general level of
activity %ae also "kmt in -the eaggjeria^atal f generation as
coE jared 1» "fiie controls*
ISie taiQ of mortelity me very h i ^ in Ifae progeny
of Ibe -tapsated individuals. Wilh 0#1 mg metepa per tOO gm
body wei#itf adiK!Mste3?©d in mter# •iie offering «ere a l l
s t i l l bom, Wilh increaetog dilationsi l&e off spaing were
nsmit
Sffeet of aetepa 0a Uie utptoaiotlir® poteatlai of Battas MSSiS^lEi
(Qieialeal atoiaisl!®»@d oially to t&msl^a only)
Mo» ^ 5 * aSHaPA Ho, of offspsti^ %iieii Io# of off^2lag ^ea aiXt*» (m/ioo m ^.¥t.) aiiuied i<iiife Ai.a)EO£« tea ii'Ss msfrtSB WAKE*
8 8
8 8
7 *• 6 6
nil BIS
wiiiiii^wirWMiw)iMii>>wMwl«liWiiiwiiiWW(lw»i^^ illwii<iii»ilw»i»»iiil»iWw«Ml»ww*rwp«i»»iwiw liiiiWwrpii»|niiiiiw<ifirtiwiai*iiMiiiw»w»ti»iWWi«i«lMPrt^^
* Data la&m^ m, -Siir e leplleatBs of ®a<ai
- ®ie aaimals died ^ ^ out 3?8pi^dacdag,
1.
2.
3,
4*
5«
6»
t. 8.
((mmyh) 0.02
0*025
0^05
0.10
0.20
O^B
0.50
N
10
9
8
7
6
5
4
3
2
1
•
, »
•
J I 1
^ ^
1 ...
S w ^ •
***s
1 1 1
• .WATER
. .ALCOHOL
<N in O <N
o o CS
in o o
o o
in
3 o (M a
i n (N o'
mg METEPA /lOOgm BODY WEIGHT (TREATMBNTTO FEMALE)
FfG.3
Pig. lY Histogram shovang the effect of metepa on the
reprod-uctive poten t ia l of Kattus noTvegicus
(treatment to females).
(rrmn
J Mlliafci
WATER AlCOHOL
bJ N 55
10-
9-
8-
T
6-
5-
4-
%•
i a • • *K
Mil
j ra MM « k mm • a
*« •• • « • 1
*«
• * • «
k k
U
" '. .< *.. '.'•
; •
• '
,'. .'
, ' , , • • ' \
. - ' •
• ,
•mJ
• • « •
»* M a
• • ft« « k
•» »• *• k > > l
*« • m • • • •
*% • t t
»> » i
f . • •
•.
*
•
• '
' !. fKii
MO Q02 Q025 0.05 OJO 020 ^ 5§0
mgMETEPA/100g»pBODY WEIGHT (TREATMENT TO FEMALE )
FlG.i.
17
b033i aliVB but ilk no case did ifeey ^rvlire t i l l maturity*
In fact none survi-TOd tjoyond tm vee^n* He si stance to
adverse factors like heat and cold seemed to he reduced in
-Qie progeny of tajeated males*
Slat eUijl alcohol also influences 1tie l i t t e r slse
of altjino ra ts i s shown in tafele 1 %ihi< clearly reflects
a distinct reduction in -fee ^ze of the l i t t e r \d"th Ihe
use of alcohol. For the i Bie ooncentratioaa, of metepa there
i s a s i^ i f lcant difference in IJie number of progeny in ihe
two sets of experiaentsf one wdllii metepa dissolved in dio«»
t i l led tiiater and -Qie ot^er wilb metepa in C jCH* Metepa
and ethyl alcohol seem to have a greater adverse effect on
the reproductive potential of ttie species concemed, ^Qiis
difference perEdsts cond-stantly in a l l Ihe different
dlluticais givmi (liable I)# Ihe l i t t e r slsse vae also affected
by administering ^^et alcohol by I tself as I s evident by
1fae ccoitrol esperiiaents «here only alcohol i as administered
to -the individuals* But Itie difference between the two
espeximental series vas apparently confined only to Ihe
difference in the nimber of progeny produced. Ho o-Qier
change was noticed^ no external or in'teixnal abnoro^itles
found eilfeer in i2ie series treated wllti metepa and distil led
water or in 'fee series treated with metepa in e t h ^ alcohol.
f AH.S I I I
Iffeot of Hetepa m ^0 l»dy i ^ t # t of effapriftg
SiiHo# Aat« ile1: pa A'vexsage « s i ^ t of Avaasag© %fei#i1; of (mg/100 gm) offspring %toea offsipfing %4ieoa
^ s t t l l e a wate» Alisolute Alcsohol Clpa) Cga>
mm'mtmiiMmtt»''''**nmmmmmminmmimiimm$immm»'^m'm»*
f geaemtlQB «- aiemlcal aaatinistepped to males pazeats cmly
4»040
4* 281
4.140
«
1*
^#
5*
4»
? f
6#
T.
8#
CoatEOl
0»02
0 . 0 ^
0.05
0,10
0,20
0,25
0 3 0
4,60
4.415
4.360
4.520
4#46
>••
tiuiv
••
f gajioxB- oa • cfaeiaioal atetnistei^ea 1» feiaale paijonte only
4*370
4*412
¥
4.386
4»3es
1*
2#
3.
4f
5,
6.
t» 0.
OontTOl
0»0g
0.0:^
0,05
0*10
0»20
0»23
0»50
4.680
4«6d|
4f
4,492
4t545
4.4T4
wm
-«»
• Uala laasefi cai -Biyoo a?@pllcat90 of oa«^.
+> •&© aaisjals died yllhomt afoproduciiig.
Hig» Y ^W Histology of ^Q tsstes
? - C<mt3pol teslaes
>
CD
KLgs TEI & nXl Hls-tology of 1he testes Wl » testes treated vdlh alcohol
YtlX • testes treated \d1h metspa and aloohol
>
>
m 18
Hhsxe m,8 Q1@O no ^mi^mn% 6itt9tmt» of ^ i ^ t of size
Taetween ^ e pxsogeay of Ih© two sBts ('SaTSlQ I I I ) •
Bis-toXogieaX sttidiea of liie tastes of 13ie t3?eat&d
msl^B yeze p^rfoxmed to stady 1ti@ cshan^s eaussdt i f aa^t
after tteataent wit i aetepa* Ho diff&teaee of histologicseX
stsactaie was olJaerwd Tjetween Uie sectlQas of tho coatiola
and -^os8 of Iho eaEporlmental miiiaias as i s ei^dent from 1tie
photogmphs (HgtiaPes T & tX)« Also no d^ffei-^nce in Ij&e
stxuol^xe of tests8 ms obgorved Ij^tiisen Hie elide a imde
tsom 1^Q aloohoX treated indiirldoals ond •mam vhicb had "been
adi!iinistQ3?od water (Hgiijees^Tri & "^XIl),
In the experiffl^ts wi-fti fecmles toot Ihsm \mQ a
eduction in -^e Xittox* &im viiti ino9?oa3lng cono^trationa
of E©t©pai S05ae ccaio^itjBatlons as 0,vmi to IJie laalea weare
used foy *ftie feiaalasji Bat in "6s© m^m of females ^0 effoot
of "tie dheiaostezllant m,B not as nmi as i t iias in ^ s males*
Olie foiaales %J©iPo appareniay mow resistant to Itie aotion of
1 0 G^aaostezilant llian l^s laalas^ In )3alesi Ihe h i # e s t
concentration %liere off coxing coisld l)s olttainad (e-vsn %ou^
s t i l l \iom) y^8 0.1 iQg* In Ihe cas@ of fsiisales siren <&ose
adainistersd wilJi 0.2 mg mtstepa p©3? 100 ga Iwdy w i ^ t
produced offspring %liidi mxe not s t i l l t)om (15atlQ II)»
- 19 *
'Sb.B effect of alcohoX (Ualal® U) w,e not as apparent l a
t^mtXee as i t m,& on siaX^s. One sffsot hoitisT^r nas oomon
in tbte pirogeny of t3?eatsd fessalss and males i#e« none of 1be
p. inaiviaualg sarTi-rod to iaatuiity#
-. 20 •
3??: ^,qi? s 3 ; QH
StexlllzatiQa as a is©-eiod for control of o^oadous
MologicaX populatians i^ laaaed on Hie mcoessfttl exacHoa'tloa
of -Qie BGf&v voisa flyt Coohllom"^^ hoatnii^a^ap (CogueseDt
from 12ie Island of Curacao, Hie Idea of inteodudag sterile
males iato a ua tu i^ population of screw idoxm f i les to
obtain control of 12i0 aspe<Ao3 v&s f i r s t propooad la 1937 ^y
aiipling (Stiipllngt 1960) p
Sterility has Taoeai defined In siany ^layst perhaps due
to mrlous causattw factors* According to KUgore (1964)
s ter i l i ty I s ^ e inalallity to produce offspring; a sterile
Indlirldual cannot contribute a genosm to tlie lOBxt generation.
!2iis I s not universally accepted Ijceause I t does not Include
individuals ii io do proaue© progeny Oilch ar^ in turn incapable
of reproduction, s ter i l i ty my be caused by various ffetctore
e.g#f InfBcundlty in females? aapesala or aparm inaetivatiott
in aalosl inability to mte or dominant leltml sutaHons in
me reproductive cells of eiifeey -gie male or Uie fesiale* ia i
Ifee^ concU:tlons lead to s ter i l i ty and can be produced by any
of -me agsnts Ihat effect ttie reprodttctlve system* Some such
causative agsat$3. are irradiatloa, cheaosterilants and oilier
antLfertility agents*
- 21 -
Sterllizatlm wllh dieialcais has, ia »©cent years
receivad ooagidssalalo attmtion Ijsoause of the possilitilty
of using Ihem to replaos X-»ray and Sp.ray irmdiatlaa for
stexHisaUon prooees* Madi of the voik has been doce oa
insect dienoaterlliaatlon, QoldgHdIh and h i s colleagues
(&oldj3ad."ei and fv&akf 1952) wojking yiUi 'promphi,'^^ «ere
the f i rs t inwetLsators to report that <^rtaia caiendoals
could <^uae 1he retardalAon of ovarian development in
inaecte resulting in the aterillsatlon of female a aeac
Almost sdmultaneoualy Mtlin and assodatea (Wtlin §ji j^,,«
1957) reported on the effect of dieadoals on -the ovarian
development of bouse flias* Woa csn ovarian inhibitors of
"bo-fc house fl ies and mosquitoes «as also done by Astsher
(1957).
'Ehe biological effects of tie alkylating agents
apparently vary wilh the agent ueedf route of adtainistarattont
Ihe dose administered as ^lell as olher factors* Also ethylene «
imino group has been introduced into a number of types of
compounds and i t s diemieal activity i s influenced by -fee
remainder of ihe molecule (Wheelerf 1962)*
During 1he preesnt stadyi m t s of both eesss weas
orally adainisteyed wllh stn^e doses of varying concentrations
of metepa, Coa^lete efficacy of -fee chemical on ^ e aniai^s
* 22
y&B peiliape not aanifestea ag dry ariadiig of a dxemostsnianl;
l a more effootle -aian ^«a addled ^ liie ,^e1; In soluHoa
(Pain-fcer aad K H ^ T O , I964) as was don© in -tii© caae, Aleo
accoafding to Salnea and Elmljoipotii (19^4) ajstepa I s ©ore
toxic to sata vHsmi given 1)y stomadi tulw in ^ t a r aolutloiasi
•ttian \iien gXvm as a oomponent In dlet# Part of Uia difference
p jr ^ dae to the aMllty of Ihe m t to dstoadfy a large
dose vfeen i t i s ingested intsisAttsniaiy duxlng an satire day
Uian \Jien i t i s gi-s^n a t cm© timSf Hot^-vert a t "Si© least a
part of iiie difference mi^ t be dae to "breakdoyn of metepa
Ijefore ingestion T>y 1fee animal (Gaines end Kimborou^t 19^4)•
Bttt| according to Wilson (i960) our knowledge of the effect of
drags on cellular processes i s raliier lizoited* I t has been
an object of dheaolherapy to des^aribe •fee nwde of action of
a epecific drug as related to i t s biological effects on
sens t t i^ cells and to desoribe in molecular detail Ihe inter*
action betwofn liio diug concerned and i t s target or receptor
02 ;an wilhtn -Sie cell (Caaeront t975)» ^ust as alkj^ating
agents inhibit proliferating oellst cells of cancerous cell
popuiationst ^ey also playha'TOo vith ^ e nornal proliferating
cell populations of Ihe host tlssttes (Oamsronf 1973) #
Stadies on the ant l fer t l l i ty effects of 8.Jcfl5.atlng
agents on laaiaiaale haw been made by %stein j j s^A 19tO)
and SaSnes and Kimborough < 1964) among others*
/
*• 23 •"
Ih© effect of metepa tan Ui© teprodttctl'7© potential
of 1Jie indiiridiiale I0 oleaiay ^oida ta -fee ZB^olts* I t i s
e^aent f em 'Qi© data Ihat Hie e fffeet M&Q rmm pronounced
in i^e case of malee itian in Hie case of f^stales* l^isaXes
treated wiHi Hie chesieal lAien aated wiHi noaaal luales ^owed
less s ter i l i ty .th»Hiat tiio^n Ijy a cross in Hie reverse direc
tion* sucli a phenomena occurs not only in mmmlB Isut has
also tjeen repoa?ted by 'miiou© yo^k^VQ in insects (Khant 1974t
Murvodbi g j jg|,*» 1964). In cags of sales eiren a s litiSle a
dose as 0,02 ag per 100 gm body ^isight «as sttffLolent to haw
a posltlim effect, lAiereas in feaales a h i ^ e r dose (0,5 mg/
lOOgm '(K5dy wei^t) iiiae req,uired to show significant poeltlire
results* these results indicate that Hie induction of
s ter i l i ty Hirou^ males i s geneially lauch more successful
Hian Hirott^ females* ITot maotk i s Icnovii about Hiis phenom ion
and Hie reascJna behind i t are more or less sj^eoolattipe.
Since Hie animals were sated after theadministration
of Hie oheaicalt i t seems probable Hiat Hie ©hemioal a i ^ t
haw ta^en effect at any s t a ^ of Hie gametogenlc process*
In isales Hie phenosienon of Gg^roato^snesis i s lengHiyt
requiring 16^20 days for i t s con^jletlGn <iralb&ndcT# 1970).
I t i s Hitts possible Hiat depending on ^an Hie mating took
place p ffliy stage from primary spermatogonia up to spesajatida
mi^ t haw been affected. I t i s possible Hiat me1@pa does not
24 »
act on -610 sponas ihomaelves <31i?eoia.y but acts on Ifee Efpera
mother eells» To "verify Uie stags in ifae apeuaatoganic
proceea a t vMdi tia ^Sieialcal actedf Hie day oa ^liSi laattng
took place \ijas obeerv^fi* OaXculatlag -eie gestation period
in fats as Iwing 21*25 daya (HaHsandovt 1970) i i t yae noted
Ifaat ^ e day counted as day 1 of paregnaaey iipae in moat a
oaaee 15*>17 days after iheatodaietmtiim of liie steallant.
I t isf Hieisefore, e-^ident -tjjat '^e sterilent acted not on
Ibe siperms Itiemaelves dixectlyt tu t on eai^ler s t a ^ e like
•ttie epe«a mo Uxor cells or primary espeiBjatogonlat oaaalng
aiangee like inhibitiQa of syalheeis or alkg/latlon of Isaac
pairs or forraatlan of cross linkages in i t s genetie material.
I t i s %iell knom ttiat i^rloas alk|3.atlng agents ^cv a
selective effect on *Bie different s'teges of speMaatogenesls
(Hayesf 1964)* According to O acksen (1959) "ttoe ellija.eneiiaiaes*
Ihe group to ^ i d i aetepa tjelongsi !»y interfe3» isdlfc fie
ftmc14oo of speroatoaoa or ttiey aay ini^rfere wllii Hie eaj^y
de^lopiacsxt^
I t i s also well knoi!© Hiat alks^atlag agents cause
s ter i l i ty "by indudag doainant letiial notations in -Sie
reprodaoti'SP© cells (LaCfeaacs sjks^»§ 1968| Ehling M;^^**
1968) • l i e l r autagealo propertlesf t»1ti mitotic as "well as
meio'acf ha'TO t«en Inirestlgatedand reported by a large number
of woalcers (Auorbaebf 1958; Itelimy and Hahmyt 1958? Hayesi 19645
HaqL0.t 1974)*
- 25 -
Uie nmlagenlc and earoinogejaie aotliritles of alk^ating
agents point to -the deoxyrilsonuclelo adda (i:®A) as an Important
site of alk^atton. der© la nudti ©^dence -feat alkylatton
of WA ooours l a ^P-"^ ®3Ld J^ - .-fty.... (Brodsss and lawloyt 196it
1965f 19651 CxBlhom and loTaertSf 19^61 Vtooelert 1962> and
-eiat Ihe most extenalve alkylattcsa ooours on 1fee N-7 8itt.t of
Ihe guanine i«sldJie8t Alfeylation of Suanin© In this position
oauses a labilisation of -&e glyoosldio linkage Isetween 1ti&
"basoand 1fae deoxy riTsoge moiety and 1he rupture of Ifcis
linkage "between iJie two leads to depusinaHon of TMk wttK
concomittant liberation of alkylated guanines (Bosst 1962)*
While mutation and oaroinogonesls m i ^ t "be due to direot
alkjiaHon of MAf i t i s quite posedble Ifeat toxicity could
result f*om interference tdiaa cellular functions \b.idi might
or might not inirol"W@ alkj/lation of WA (Wheelert 1967) •
Among sudh functions are ^nl^esis of Mkt MA and protsine*
25ata obtained (Cxa13Tiozsi and Robertst 1966) have sho\ia Iftat
inhibition of sjmthests of nucleic adds i s a commonly
observed effect of alk^aatlng agents and tliat "the inhibition
of synlhesle of MA i s usually greatsr than Ihe inhibition
of BHAf .Any of Ihe above mentioned factors may cause dominant
lelhal mutations in the reproductlvs cells thus causiiig
sterility* I t vas no tad in oaes of o-eier alkjilatlng agents
like tretamine end busulfian too, that tiQ chemical i n t e r^ re s
vilh Ihe erpezmatogenie prot^ss a t "tbe spQima-togonlal stage
ana cattaes a ^igjit leduottoa In aerom goamdotrophin (Eari
Kamboj and Harl^diandaiaf 19661)«
In eaee of feaalest alkgating agents oauge s ter l l t ty
by tnJiiblttns fecundity, Hfeie has Iseen observed bolti in
a3J£j^atlng agents and antlffietaboHtee (IiaChanee ^ B^f^ t968).
I t vouldy Hiersforst be Itiat less nombe? of ova ihspa nmsl
are produced and rea<di laatttrity and a3?e ferttlisied or i t
coiad also be -fiiat fetuses wco resorbed after iioplanl^tlon,
Ihe laaadLmum tsifttogsjaio aotton of antliaatabolltes was noted
to be between a»l3 days of implantation (Vidinyakovi 1968)
and for a l k ^ t l n g agents during Hie implantation of embryos
on Ifee 6-ei dff.y of pxsgaancy (Aleksondrovt 1968) t probably doe
to intensive lostaboXi^ in -tie embryo during liiis periods
Primary mo^rphogenetlo reaotlons to aUcj/lating agents are
manifested In lahlbltlcn of prollfiezating oeiters (cytostatic
effect) or dea- i of cells (cytotoadc effect) in mltotleally *
inactive but no!n'«*differentlatBd aonest \Mcfe I s of basic
in^ortaace for teratogens sis (Alekaandrort 19660.
Ihe la^ of any histologloal damage to ihe testes may
be at^Rlbuted to lhe ^ c t -that ihe ocmcentratlons \^re toe
low to cause any visible histological damage to tie tlsi^e*
I t could also be due to spontaneous reversibility of
testicular atrophy^ i f any* fbis assumptlcm dravs support
* 27
from Ihe eaqperiinaiital otjsermttoaas of Gains a and Eimtwapough
(19^4)• ^ e y obaeyrod iiiat a h i ^ fl^gxee of spontaneous
^everstMllty of ecaf^lete iaicapQ8<sopie tetotleular atroifey was
produced In rats trsated « 1 ^ h i ^ dietary levels of metepat
as -Sie lengl^ of time lietween trealsasnt and eaiaiQination
ino3?0ased« Aooording to ^emt under sosie conditions ra ts
are alflte to recover from complete testicular atrophy caused
by metspaf but recovery occurs eioiilly a t a dosage level cf
1000 ppm knova to be lasQge enou^ to k i l l some xats* Xn ihe
present study sinos 1be ra t s vers treated \d1h lov» sdngle
doses of sietepa and a consdlderatle period (40 days) had
elapsed between Hie time of examination and treatasentf i t i s
a l l the more possible that sp<3ntaneous reversibility occarred»
Alcohol i s i3an*s original "teanguiliging drug and has
been in uggige eince a long time* Over the l a s t several yearst
sdent i sts have been conducting sj^rimsnts Into the relation*
diips between alcohol a ad virtaally ©very aspect of mammalian
physiology. In addition to having an effect on iStkB reproduo-
tlve potential I alcohol al i^ affeots various oltier organs*
ihe most important being Ihe liver and the heart, freata^it
alcohol slgniflcaniay increases 'fee act ivi t ies of hepatic
pentobarbital aad benjspyrene hydrojddase in ra t s (Bubln and
liieberi 1969), !!!hese voikers have also shown a number of
-. 28
SHianoi pifoaotes accMsaaUoa of liats and elwates
1ti0 l e w i s of lalo6& l ipi4 ta?lglyc0add«s CitoMja ana Melsea?*
1969)» I t a3.8& has a clii?©ct -toxio aet lm on Uis hsart#
Blr©ot evidence of "toe aaasagiag efiset of gajall amoteita of
aicohia on heart; isasfii^ in fat© «as pxesaalBd Isy S0®aX
(I9t4)*
%e eflfeot of alcohol on litiser size %ias a ta^ td mry
early % A^es MvSm <1930)» ®ie littaar T»®8 found to h&m
decx^ased af^x* 'dm teeuismk% Warn "^m. not tm^ of vfoiSs
lias Ijeen done on "fee effect of a l^^ol en Itoe repj*odttctl^«
potential #t m t s . Con tinned atainieteation of aloohol by
gahc^taenous injections to -^smitsSi smle mtoe has Iseen kjaom,
to inereaee pxenatal laortaHty^ Ui«s deoieeadJis l i t t e r slge (Utihat 1950)* Only parental sAoe liBm treated in "til
Hi© feaales and offaipJlng of aOTengu^eequent ge^ieaatioiis
reaainlng untreated* In a l l gmemiicsia follotdng -Sie treaV
mentf l i t t e r sdee deoreasedf an effect a^aAfees to pr©na*tel
!aiat s ln^e doaes of orally aflsdnlstared alt^hol too,
imrm an effect on itoe l i t t e r Am i s evideat f!?oia -fee data*
ISie remi t s also show Itiat %tille aalee are aensttiim to
- 29 **
alecjhol tiJea'taeatt female xats appease ralber 3?©slotaiit to
lt» Bofe .\4i0n alcohol iitaa adEdajtsteafed by it«elfp as in
otmtroliSf and in comMnatLon with sietepat liie males s ioided
a stgaificant irafiatton in Uie nttmt»r of young ones per
l i t t e r ae «»iapaped to ^ e other mtiee of e3Q)erii!ients ^ki&ve
no alcohol %ia8 atalnistered im.0itQ 11)*
In case of feoalest alcohol nei-ftier hy i t se l f nor
in comMnaticai with metepa appeared to hav© any effect on
the 3?epa?odmctiTO potential of mta (Hgua?e !?># In either
case no degsneratians or other aoar|*iologlcal abaorsaalitlea
asertfealSle to the treatnent mx^ ohtained,
ih^ differential effect could he due to differential
msceptlhility of X and f chromoeojaee to e-ftianol. I f one
asEMaes that t dixomosome i s tsisceptlhle to alcohol treats
aent %lhile X chromosoae i© nott the descrepancy in the results
ohtalnea on treating males and femlea i s ©ssplained, Chromo-
soisal ahherations produced in qpeiaas cd^it he one of l&e
causattv© factors. C&romosoEial h re^s are knowa to he produced
in insects hy alcohol treataant (Midmelis and Bleggrt 1963)»
and i t i s posaihle that they are produced in trnmaln too»
Ho\^wrf laudi more experimental WOI3E will have to he done to
verify the presKit aaggeatlon and i t i s rather too early to
say anything \dth certainty regarding the differential effect
of alcohol on -me two aexss of B^t^Q Wii^^Af^S*
• 30 ••
1. I» laales, coaoentmtloas of ae-fcepa hi^ea? lihan 0.1 mg/
iower eoacentmtlanQ reaace Hie littiBr slz© vl^out totally
affecting 'Sio sraproduotlve potealiBl.
2» fh© feasle r a t s appeal? move x^s le taa t than, the s a l e s to
"tafeataeat of lastepat stace in liioi^ c a ^ cGaaceatratlaBB up to
0,20 ng/lOO ^ Iwdy i«si |^ t do a o t completely iiahilatt ^ e
rsp3?oaaetlV0 potent ia l*
5t Ho apparent a lmoiaa l i t t e s eiUier norphological o r
anatoaieal om caused ia Ihe pro^say of eilher Ibe treated
males or treated feiaales.
4« Ho hietologlQ ^aoage ttiaa noticed in Ihe tests@ of
tde treated male ra ts m&a a t the h i ^ e e t conoentrEtlon of
me tepa*
5* In ^ e DBle ra ts effect of metepa i e greater «h^
administered in comMnatim \dth Q-fesfll alct^ol than ^ e n
administered in comMnatlon \d1h diatllled \a ter indicating
ihat e t h ^ alcohol by i taelf also has an effect on ^e
reproductive potential of alMno ratst
6# In -flie female m t s -^eTe i s no diange in the effect
of metspa vhen aamlnlstered either in alcohol or in mter .
«• 3t •"
Bie effieoijs of drugs and o1fe©r dieiatcaXs to «fei<li
anlaals ai?e ©sposed el-eier fi0lit)emtely or madFerteatly
csonsitltate a field of major ImpojftanoQ in I3ie study of
MoXogy Oil da aids in ttie control of vertebjreite posts*
In -ftie present woric 1he 0;tfeot of aetepaf an alkyla-
ting agent, on ttio reprodnetlvo potantlal of aXljlno ratst
Sfi t,%? fiqiniyi^glms ¥&e studied^ Uetspa waaaaialaaistored to
Ihe animals bolti in comMnation vdlli disti l led mter as \iisll
as «i"8i absolute alcohi^ in ordter to assess Itio effect of
aloc^ol itself*
Ihe aniaals iiere oasally fed id-Si single doses of Hie
cheoloal after v^idi noxisaal feeding ^ e restamed, Bo'th males
and t e a s e s were tireated after wM<ai tliey were uated «i1ii
norcmlf untreated aniaals of Ihe opposite sex* Seiren different
ooacentrations of metepa ranging fieora 0,02 ag/tOO gm toody
^ i ^ t upto 0,50 sg/lOO ga body woi^t were administered*
Metepa was observed to reduce tbe number of progeny
per l i t t e r boifa vdien adadnistored to sales as well as to
females but -Qie effect was more pronotmcsed ttirou^ males.
"» 32 I"
Xn oass of stale ra ts Hils effeot vas enhanoid \hm^ the
c oEiicaX me fed to ©)mMnatl<ai wlih alcohol. In foaales
alcohol tey iljaelf aeaiaod to h&rQ no effect on Ifao s^productlve
potential. Ihe diffesenttal effect of alcohol on tie two aeaQSa
has Ibeen disoixeaed*
Histological etudlee of m© IsemiseA aal© teste© were
also perfoaraied to note -eie daimgBi i f aayt to ijie teetea. Uo
apparent change in "Sie histology %®s noted*
tffiio alkja-atlng agent i s thought to have Twought ahotit
•the reduction in the nuaher of progeny Ijy oauging doiainant
le-feal autatlons of Ifee reproductive c»ll9» prohaTaly in -fee
early atages of epeismtogcsiesl s. Several oiher ©ediaaissie
regarding Ifee effect of laetepa on the reproductive potential
ot MWU m:m^^S ^ ^ ^«3» aiscueaed.
•• 33 *•
• Alekeandapov ?.A» 1968t ffezatogenous effect of antitmnojwus
alk^^atSng agezitd (oaiaystosia pund^tiiamt tritoat
aplestzlsff f l ^ i Mrdf asiMUan and laaassal) ABECR ^A!i!
GiSfOL i^HIOI, 54(6) 69-82 (Eag. Sam.)*
Aectiert K*E,s* 1957# Pie- maticQi of oidpoaitlQa in the houssfLy
ihrou^ taygal emtaot ageats* Sdeaoe, 125» 938.
Auertiachf 0* 1958« Badiocdsietto guit»dt&noeQ» Badiat 138«» 9>
55-47*
Bluhfflf Agnea* S30* In^cod genatie ttmn^a* %e ieaetlos
of -Qi© mottsQ Ijy Haas Gxuaotes^*
Boxko'vaOf A*B«t i^^i Chin Chang and A*M, LimTsus * 1964* Effect
of j»B on sterUiaing activity of tapaand laetepa in
house fLyt J . Econ* Entoaol. 57* 8t5* l8»
Bwofcesf P. andP»B, Lautey, 196% At^dio dlasooiatlQa of
7s9 dialkjl guanine and i t a poaattle ijelatton to
sutag^sic propertlea of alkjaatlng agents* 5a1»T©
(London) 192s 1081,
Brookest P« and P*B« I.8\dey# 1963* Bje action of alkj/latlng
ag^its on Wk in relation to Mologieal affects of
•the alkylating agents. B^» Cell Hegoar(^ (suppl* 9)
512-520,
Sroofces, ?• a»d P*3)« Laidey* 1965* MoXeoalar median! aa of
ihe oytol^sic acttcoi of m-imeUmsl aik^atijag agents
and of 3?0sl8'tenca to Ifeis aotttm, Katua?© ittmid^m) 2561
feaeaKait 1«L» 19t3» i» S^ga and 1fee cell oyd>e ©<l» A,a, Zlssaeisaaaf &»M» y a ^ i i a aad I.l*# G^ssroa* Aoad# Prass,
Oattaiteichf B»S, I9§t» Indaottoa of tasanglocatloas in adoi
liy trieltijlanamelaajlii©* Kal3«?e (Iioadcaa) %QOi 1364»^5#
Oiamteaiaia, tf#f# and B,W» Hamilton* 1964* Ab^rptioat
exc3?etloa and at-tetjollsa of f lalselled i^tepa by
Bornvm^Tm md stabla fli©a» J« Boon. Satoaol, 3t$
800-80%
a^an a i n Chsaag and A.B» Bojkovee* 1964. qe^aitttatlve affects
of tepa» fiietapa and aphOlata on si^Hligatioa of laale
housefLies. J# So«m, ait0mol# 5ts 488»90#
Craigf A,W*t B»w» fox and H» (Tacacsoa* t9!58» Sensltlinty of
tfee ^matogemlo ps^oceas in Ifae ra t to mdloialffietio
daniga and 1-mys. Ha1»»g (Imdon) 181* 15!^354»
Om^ornt A#B* and 3*«I. Bobartst 1968* Hedianl^ of Hia oyto^
t»sle action of alkjlatliag agents in BSBCimllan oella
sad euddaxoi for 12i© reaotal of^Oct^eted group a foisa
UKAt KatU3?© (Iiondcm) 211* 150»
- 35 *
Bame» 1)#A* and C.H» sshialdt* 1964» Uptake of metepa and
I t s efl^cts on 2 lEfpades of mosqtiltoea and house
fliost J . Econ* Batomol 57$ 77-0t»
Mlingi, tJiH,, B»B* Cuiaingf H,?# Mailing, 1968* Indactl<m
of doiaiaant leticCL mutations "by alk^atlng agents
In male jade©. JIat. Bes. 5(3)* 4l7-428#
• Bpstslnt S,Sft, Jtotoldf E., Sipeinlaexgi K#» liaddLnto^, I)«,
^lameri H. and Bi^opt X* 1970, Ifeitagenlc and antl*-
fer t l l l ty effect© of topa and me-tepa in ad.ce# -' . '
mum* AEK. mAsmmh i7(i)t 2>40,
Sahmy» 0,G# and M»J* Bahmy, 1958» Dilecus^on of paper
<t!tttagenio effects of a lk^at tng agents" Cby C. A«eav
l3a<di), inn, I.X» Acad. SdL* 68: 756«748.
l9x» B*w«| H. Jadcsont A.W. Craig and ^H^ Glover* 1965*
Effect of alkylating agents m, spe^aatog^aesle in
tie ra1)Mt« J , Bepisjd, Berfell* 5s I5«2t»
Gaines, (?.B, and fi,B. Hiaborou^, 1964# foaddlty of metepa
to 3Jats. a«Ll. md, Hllfc. Ong, 31, 757-745»
Goldanl.*ait E»B, and J . Smnk, 1952* Steslli ty in -fee fensale t m i t Hy Sroaophila melanoga^qt r produced lay -foe
feedins of a folio acid antagoi:dat» AiMyioan J . Phyaiol,
171(3)1 726-727*
**• J / O ••'•*
0ou<lif H»E, 1964. Chemosteriliaaiiim of houaeflles hy
"tepalsiiiit ia pup^ stages* J» E&s&* SatoiaotSTt
Hat^t S#f,H«# 19?6» Stadlts <si t ie effect of Ibio^pa ©a •tie iKme Borrow ciiiJoaosomest liW3? and t i s tes of
alMao mt8# M» Phil* tosstrlstltm A,M*tJ»
Ha5©Sf W«jr, Jr» 1964. fhe toadoology of ^©mosljerllants*
Ball, ma, HIHi, Org, 31# 721-736,
• Hendrj, J.A#t E*F» Hos»srt f*'i** ' 9® and A#Ii» ¥alpole« 1951*
Cytoto^o ageats HI* B$:ilifa1iw@ of e-iiyleaiadnes.
Brlt# Urn PMCTac50l» 6i S57-410#
*Ja<a£g«aj» H* I959t Aatlfeytlllty ^iJa-tjaaoea* flmssmeol^
^ i r . . Its 155*172*
KaTf iUffitjai Bti V»B# Kaml>oJ and Hail iSK&andTiat 1968» Ef^et of $00^ dieiaioals on s|»e;iiia1 @ ei@ 6 in I h e ^ s Acmkej*
J* Itepifod, I^Ttai. I6<2)i 165*170.
Wtkmit %tl» 1974# sisidits (m Hie g^e t lo sffoots ofcfaeaoateillaat
I ^iotspSf astepa and hompa oa Hie Mologf of j^p^
yioeaf .ffe lf gai s Wol^teim, ]?h.»» 1fe«rf.o A»E,fp
Kilgoa?9t M*W», 1964, in Post Om^rot od, tjy W.W, Eilgoi?© &
Dotttt.
Inlpllngi l*f# I960, fimdiisa-ttKm of so20¥ %©aa flLir, Sd..
Asioy, 20^ 4) s 54*61,
I,aByeeqttei &»Ct t96l# Studios wlti ^ ree alk^ilstlag agoats
as house H r otoillants* J« Eooa. I^toaol* 54{4)t 684* 89.
*. 37 *•
LaChaacst %*'&*§ B.f- Horlfa and w.K^asaea, 196®. Oy1»g^a0tie
sad ceXlular Ijasls of dJtialcally iadaoea s tor i l i ty ia
i n l e t s in { r la^p lss of insect diemos"terlXi2atlcai
8d» l3j I.a B3?eeta® S«0» aad ad-Si*
admsl i s f Ai end iU ll©®By» 1965# la-temdtloia of cHiroaaldLd toseaks infiaeed toy 1lii?e6 m.ttetmi% mdloialmetlo compoimd©
Ha1ai3?6 (LoJidcai) 199« 1014»lOl5t
Miiaiiii I.# B*A, Btttt md f.J , aiorHno* 195t. Effect of
lall^tlc pois^ma <m hows© Hi" eviposlttoa# Bhya, Zool*
30(2)8 133^136.
Murvo^t C.ll*f I.aBi?@oq]tt0» &»0. jsad Saiiliii C*I. 19S4» Effects
of Ifes^e dxefflostesilaats oa housefly loagQidtj and
s te i l l l ty . J,Eocsi» aitosol'57» 89-9 3#
Hsateidovt A*?, 1970. ]fep3?odU0tiTO Fhyslology, D,B. fam-
poxeTOla So»s & C # Fvt, I.td#
la ia ter t E^E. and W»W« Ellg02©i S64« topomz^y and paiaanent
©thrillsatloa of hoaaefXles ^*Si ctianosterllants,
J . Bcon» aitoi9l-57 (1)3 I54-157.
PlngieiKtl^to^mirtiy and Baimst sM*, 1967* Ifeta* food g»a4»
!I!0<toolO|^sts xi^seadi as^^oia-ilon of Isidla*
3?la|^t f«W,i W«S. Itiptajt <l»A»f(lia ]aan and#*M, Mdy t9&2*
MataT^olisa of s»tap«aida in a ^ s ^ t o a t , f l ies mS. sHoa,
^r,Bcoa*Eatoa9ld5« 607-.t3if
• » 5 ^
in mm. @nd m t luaiuc-ytcm azid iiili|.Mtlaii li r eltiaaftl*
Sig^ 19T4 fapoa Meaieal mm&it^ xepoarfef •* Ifeat l a ^ e effeot
« iriiiii|a}£0Yf li«S« t968« Bsoper^di dt '&& ^easagiag effect of
6*»Hte3?»ptoptxflae during diffferent sisges of ra t
0ma^yo gues t s . fMMOlL fOKStEOIi 31 (4)t 460-481»
lie'&ieafbetf B*K, 1964. fapom Fest Conti^lt MologimX phyrf-oal md selected {^tsAoal meHiod©* ®d» Illgora W»E* and
f e e l e r , §»?# 1962* Stages 2?elat@d to tfet i2e#ianlssa of
aetton of cytoto^do aXk gating agmta* 1 ae-^0¥#
Gaac^r Bes. 22f 651-680*
toeler, §.B. 1967. ^e^vecfc^ Pn,oa^^^ *
Mlstm, ©#3, I960* lie study of drug effects a t oytoXogioal
Xewl, Ittt. Be^. Cytol, 98 a9>^)4»
• asfefsmceas aot coag»a.t9d in oal^toal*
