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Discussion
DISCUSSION
In this study, two types of stress were presented to the female rats aged
between postnatal D5 to D70 Although stress IS presentable from day zero, a pllot
study resuited In some mortality of pups when stress was presented soon after
blrth Mortality was also hlgh when electrical shock or any other stress was
presented at an early postnatal age However, maternal deprtvatlon was found to
be an appropriate stressor ~n preweanlng rat pups Maternal deprtvatlon 1s known
to decrease ornlthlne decarboxylase (ODC) a sensltlve lndlcator of stress effect
Changes In ODC due to maternal deprlvatlon 1s dtrectly assocrated wlth the removal
of actlve motherlng behavlour and not secondary to malnutntlon or perturbations In
body temperature (Kuhn et al, 1978) Present observations lndlcated that after
each perlods of maternal separation when the pups were returned to dam, they
sucked for longer per~ods and the dam groomed the pups longer
The schedule for maternal deprlvatlon stress and foot-shock stress was
planned ~n such a way that nelther the subjects reached a breaklng polnt for any
cond~t~onrng nor hab~tuat~on could occur Rosenfeld et al (1992) have shown that
repeated maternal deprrvatlon stress does not have cumulated effect nor the effect
of stress was add~tlve Slnce repeated short-term stress was presented on each
day and tts effect IS not llkely to be addlt~ve as descnbed by Rosenfeld et al (1992),
the present study when vlewed according to thls, ~t can be considered as acute
stressor However, on the other hand, aggregated effect of maternal deprlvatlon
from 95 to T?O anp' f;,-!-sho:i ,.:rest. fron~ 921 to D70 deserves to be designed as
chronic stress. Short periods of maternal deprivation do not have cumulative effect,
however, there IS a critlcal length of deprivation beyond which persistent changes
ensue adrenocortical responsiveness (Rosenfeld et al, 1992)
In the postweaning perlod, scrambled foot-shock was given with increasing
intensities, so that. tolerance to shock and habituation could not take place. It was
noteworthy that immediately after the foot-shock session the rats returned to the
cages consumed plenty of water but did not take food which was available, Instead
they slept for sometime Explanatron to this behavioural change may be due to the
fact that during the shock presentatlon the rats urlnated and defecated several
tlmes causing loss of fluid. So they replen~shed the fluid loss. On the other hand,
instead of taklng food they slept, it may be because during shock presentatlon the
rats performed greater amount of physlcal activrty which necessitated longer perlod
of rest
From D71 to D100, although the rats of the post-stressed group showed
greater percentage of bodyweight gain, the weight of body, thymus and ovary
remalned signrficantly lower lndlcatlng very l~ttle recovery withln a perlod of 30 days
Although Rosenfeld (1992) stated that there IS no cumulative effect of maternal
deprivation, present observations shows that chronlc stress resulted in sustained
effect atleast for considerable period.
Through hypothalamo-hypophysio-adrenocortical axis, stress causes rlse of
ACTH (Kant et al, 1992; Anderson et al, 1996), cort~costerone (Raab et al, 1986,
Baez et al, 1996), prolactin (Rosenfeld et al, 1992; Kant et al, 1992). It is also well
known that strgss brlngs about alteration In biochemical substances l~ke
glu~0corticoids, serum cholesterol and trlglycerldes Short perlods of maternal
deprivation durlng D5 to D7 d ~ d not affect the triglycerlde level much Observation
on D7 suggests that stress effect was not seen initially or the mlld reductlon may be
due to acute stress. as Robertson and Smith (1976) have reported that acute stress
caused decrease In trrglycerlde level Though maternal deprlvatlon IS considered to
be a milder form of stress. ~ t s chronic effect durlng llnd and lllrd week of
preweanlng period of rats llfe resulted in elevatlon of triglyceride levels Further
during the prolonged perlod of foot-shock stress the level of serum trlglycerlde
rernalned persistently hlgh due to chronic stress
Stresses llke cold, heat foot-shock and social stresses when applled durlng
pregnancy, resulted In lowered blrthweight of the offsprings (Salgado et al, 1977,
Pollard 1984) Postnatally maternal depr~vatlon d ~ d not show early effect on the
bodywelght of rat pups But the effect could be seen by end of third week Thls
notable decrease in the percentage of body welght of stressed rats might be sequel
of decreased serum growth hormone (GH) concentration Kuhn et al (1978) have
shown that wh~le maternal depr~vatlon stress causes elevatlon of ACTH and
prolactln levels, t decreases ornith~ne decarboxylase activlty In braln and heart
muscle and serum GH concentration In the present study the decrease In
bodywelght may be attributed to decreased GH level due to stress That can be
supported In the reductlon of cytoplasmic volume of somatotrophs due to foot-shock
stress (Mukerjee, 1987)
In an earller report, Dess et al (1989) have stated that foot-shock does not
i L.U; -ess t",? !)ody wel?ht and ~ ts effect can be attenuated by control r,.
shock In disagreement alth thrs, In the present study ~t was observed that foot-
shock shows lmmedlate effect on the bodywelght by s~gn~f~cantly retarding the
bodywelght gain In growlng rats Here. ~t may be polnted out that the srgnlflcant
effect on the stress on bodywelght observed ~n postweanlng per~od may be
cumulative effect of maternal deprlvat~on Retarded bodywe~ght galn can also be
attributed to excessive phys~cal actlvlty ~n the shuttle box and Increased per~od of
sleep and poss~ble decreased food Intake Stress has been found to both Increase
and decrease food Intake In anlmals (Rowland et al, 1976, Donohoe et al, 1987,
Dess et al, 1989) as well as human (Wlllenbrlng et al, 1986, Grunberg et al, 1992)
depending on the stress rnan~pulat~on Decreased consumption of food and
Increased Intake of water leads to reduction ~n the body welght has been observed
~n rats exposed to hlgh amblent temperature (Narendranath and Klracofe, 1976)
and housed ~n crowded condlt~ons (Gamello et al, 1986)
Chromc stress Induced decrease ~n the body welght whlch IS revers~ble
w~thln 14 days of w~thdrawal of stress (Anderson et al, 1996), whereas ~n the
present observations, 20 days after w~thdrawal of stress and restoration of normal
amb~ent cond~t~ons the s~gn~flcantly reduced bodywe~ght reversed towards the body
welght of normal rats
Thymus gland changes ~ t s slze wlth age of the growlng rat Nevertheless, ~t
IS not strlctly akln to change w~th we~ght of the body ~n growlng rats Thymus welght
Increases In neutral envlronment and ~n cold envlronment ~t decreases along wlth
the bodywerght (Hale et al, 1959) In the present work thymus of control female rats
growlng In neutral environment have been found to Increase steadlly from DO to
D56 Present study also revealed that In stressed rats percentage of thymus In
relation to body werght has reduced Reports of Mahmoud et al (1994) suggests
that wh~le contrnuous l~ght for three generatrons In mrce brlngs about thymlc werght
loss, contrnuous darkness results In thymrc werght galn The present results
rndrcated that wrth normal L D cycle, thymlc we~ght loss was observed showrng the
effect of electr~cal foot-shock stress actrng as a strong stressor
Sex and specles d~fference may have recognizable bearrng on the
morphology of an organ Earlrer reports on thym~c morphology (Metcalf. 1966,
Srmpson, 1973, Ohtakl, 1988) have shown that female thymus 1s heavler than the
male thymus Thrs IS not comparable wlth the present study because thrs study IS
exclusrvely based on female wrstar rat thymus
Exogenous cortlcosterone treatment was shown to reduce thymrc Index
werght (Forsberg, 1995) Stress Induced by inject~on of ACTH, cortrcosterone
(Oksanen, 1971), hydrocortrsone (Lee and Domm 1964), adrenal glucocort~co~ds
(Sobhon and Jrrasattham, 1974) and gonadotroprns (Hammar, 1936) enhances the
tlme and rate of ~nvolut~on Stress rnduced, Increased cortrcosterone as shown to
reduce thymrc werght and cause lnvolutron Drastrc thymrc welght loss has been
reported after X-ray lrradlatlon (Gregorre. 1943), but ~ ts resemblance wrth foot-shock
stress IS debatable
In growrng perlod, Increase rn the thymrc werght does not parallel wrth the
bodywerght, Axelrad and Van Ded Gaag (1962) found that Increase In the thymlc
welght is faster than Increase In the body we~ght Similar phenomenon was clearly
observed In first 5 weeks of rats postnatal lrfe In the present study While we~ght of
thymus In mice is reported to have reached maxlmum at the age of 40 days to
follow a steady decrease thereafter (Endo and Kanyama, 1998), in rats of the
present study the we~ght of thymus reached ~ t s maxlmum on D56 and continued to
decllne thereafter
In female rats, thymus we~ghs 7 55 mg at birth whlch is 0 1759% of body
welght (4 29 gm) Interestingly, thymus 1s an organ wh~ch progresses In welght with
age After attainment of maturity its regresslon of thymlc we~ght followed the
advanc~ng age, what is termed as 'INVOLUTION' After blrth thymus has shown to
Increase In srze at a galloping speed The welght of thymus reaches its zenith on
D56 thereafter the progression In welght of thymus changes to regresslon (Table 2)
in observation showlng that rate of progression In a thymic welght is faster than the
rate of regresslon At the turning point between the two sequences there 1s marked
retardat~on observed around 56 to 63 day The turnlng polnt of sequences 1s
specles dependent In mlce (Metcalf, 1964) the involution began at 6 weeks
whereas In the present series of observat~on, ~nvolutlon followed after gth week In
control rats Thls can be confirmed by percentage growth and regression In relation
to bodyweight Thymic involutlon In humans 1s not related to puberty alone
(Stelnmann et al, 1985) Gradual decllne in homeostatic potentla1 that characterize
the aglng process can be tr~ggered by early involutlon of thymus (Goya et al, 1999)
Since, onset of puberty is not manifested at a fixed polnt of tlme, its direct relation
with thymic involut~on would remaln a probability In relation to time
Present exper~ments have shown that stress has marked effect on the
thymus of rats Growing stressed rat thymus lnvolute at an early age Not only the
ln~Ol~t10n was edvanced by 2 weeks, the involut~on was greater than control
animals Hammar in 1905 reported lnvolutlon of thymus 1s replaced by ad~pose
tlssue In lts lobes Much later, Oksanen (1971) described that involution In rat
subjected to varlous klnd of stressors had followed by format~on of mult~locular
brown fat In thls study, on young female rats neither monolocular nor mult~locular
fat was observed Instead, In stressed rats thicker bands of connectlve tlssue was
observed along the lnterlobular septl
Dominquez-Gerpe and Rey-Mendez (1998) has pointed that age sex and
straln of rat lndlvidually or In comblnat~on modulate the effect of stress causlng
lnvolutlon of thymus Present work can vouch support from Selye who expressed
that premature involution of thymus can be associated wlth stress due to dlsease or
oversecretion of adrenocorticotropic hormone A deflclency of elther adrenal
cortlcal hormones or sex homlones In an animal, brought about by removlng the
endocrlne glands may be assoc~ated wlth hypertrophy of the thymus or fallure of
lnvolutlon of thymus
Flnally, it 1s infered that thymic lnvolutlon IS not only a slmple shrinkage of
thymus but rather than the result of a series of compensatory mechanism among
different cell population (Quagllno et al, 1998)
Total thymlc mass is constituted by the volume of cortex and medulla In
addltlon to lnterlobular connectlve tissue Reference to ~nvolutlon, the changes In
absolute volume are In parallel with mean volume of thymus and ~ ts lnvolutlon The
absolute volume has much bearlng on the volume of cortex and medulla The slze
of cortex and medulla of the thymus are affected differently due to stress or toxlclty
(Maurer et al, 1990) The two reglons of thymus are not affected to the same extent
(Mahmoud et at, 1994) HIS reports explained that, continuous llght resulted in
thymlc welght loss and the reductlon ~n volume was greater ~n cortex than In
medulla Continuous darkness resulted In enlargement of thymus and the Increase
In volume was greater In the medulla than In cortex Reduced cortex medulla ratlo
was observed in testosterone treated rats The lowerlng of cortex medulla ratlo was
due to reductlon of thymlc cortlcal size whereas the medullary slze appeared less
affected Sobhon and J~rasattham (1974) described that testosterone and
oestrogen is responsible for depletion and destruction of lymphocytes In the thymic
cortex, not In the medulla The observation of the present study shows that, the
thymlc cortex was affected by stress, whereas dur~ng the same per~od thymlc
medulla was not affected by stress to the same magnitude After prolonged penod
of stress presentation, the medulla showed the same effect The present study
corroborates wth the flndlngs of Sobhon and Jlrasattham (1974), Maurer et al
(1990) and Mahmoud (1994)
Stress caused marked effect on the absolute volume of thymlc cortex,
whereas thls effect showed a delayed onset In thymlc medulla till D42 PN As a
result of stress, the percentage reductlon In absolute volume of cortex and medulla
was slower In the earlier part but from onset of puberty, the percentage difference
between the two groups appeared expedited It may be that from puberty onwards
rate Of lnvolutlon became faster and act~vlty of mmune system rn~ght change
accordingly
Dally Inject~on of ACTH results In reductlon In the wldth of the cortex wh~le
adrenalectomy caused thlcken~ng of the thymlc cortex (Lee and Domm, 1964)
SlgnlffCant reductlon In thym~c cortex In the present work lndlcates poss~ble rlse In
ACTH level due to stress presentation Stress Induced over lnvolutlon may have
some charactenstlcs of the effect of thymectomy Ohtakl (1988) has shown that
thymectomy resulted In marked atrophy of gonads In the present study reductlon
In ovarlan we~ght colnc~des wlth thymlc lnvolutlon Present flndlng can substantlate
the flndlngs of Grossman (1985) who has stated that removal of thymus resulted In
dysgenes~s of ovary a condltlon characterized by lnfllteratlon of the follicles a
decl~ne In the oocyte number and lnterstlt~al cell hypertrophy Slmllar to the effect of
thyrnectomy (M~chael 1983) stress In Intact rat poss~bly decreases the release of
thymosln to trlgger GnRH secretion from hypothalamus and hamper the release of
LH from pltu~tary to cause dysgenes~s of the ovarlan foll~cles
The observation of present study shows that Increase In thymlc welght IS
concurrent w~th Increase In the number of lymphocytes whlch can substantlate the
flndlngs of Ball (1963) who has stated that Increase In thymus werght was due to
actlvlty of an lmrn~grant prollferatlng cell populat~on and ~ t s progeny
Thymolymphatlc system undergoes marked thym~c atrophy In mlce under
constant llght Thls IS evidenced by decrease In number of lymphocytes and
ep~thellal cells (Mahmoud et al 1994) Absolute number of the thymocytes showed
an anthmatlc progresslon w~th age Stress retards th~s progresslon early In cortex
and later In medulla of thymus However, rn the later part of the present study,
absolute number of thym~c ret~cular cells both In cortex and medulla IS s~gn~f~cantly
affected by stress In consonance wrth process of ~nvolut~on, the absolute number
of the lymphocytes of stressed rats started decl~nrng two weeks earher than the
lymphocytes of normal control rats Thymlc changes were clearly ev~denced by
lnvolut~on and s~multaneous fall In absolute number of lymphocytes and reticular
cells These flnd~ngs corroborates the flndlngs of S~mpson (1974) who had stated
that prolonged reduct~on In thymus we~ght IS due to fall In the lymphocyte content of
the gland
Stress Induced Intact young rats havlng Intact thymus, suffer reduced level
of thymosln by way of decreased GnRH causes lowerlng of pltultary LH ultimately
affects the ovary
An~mals sens~trve to l~ght dark cycle, show a var~ety of changes In the ovary
Continuous l~ght reduces the size of the ovary (Pomerat, 1942), on the other hand
prnealectomy or cont~nuous darkness causes reductron In the srze or welght of the
ovary (Pomerat, 1942), Relter, 1968, Hoffman and Melvin, 1974, Loudan et al,
1998)
Slnce there are confllctlng reports on the llght and dark cycle or atleast w~de
vanat~on has been reported In the ovarlan actlv~ty of rats, the present work was
conducted at equal l~ght dark cycle (UD 12 12 hr) So that effect of stress may be
exclusively the effect of maternal depnvatron and foot-shock
It IS documented (Clarke and Kennedy, 1967) that In voles, ovaries welgh
more In laboratory controlled summer s~mulatron than In laboratory simulated winter
On the contrary, mlce reproduce when kept at sub-zero temperature (-3") the
reproductive organs do not regress under cond~t~ons of low envfronmental
temperature (Barnen, 1956) H~therto, there are confllctlng reports about the
amblent laboratory temperature To stnke a balance between the two extremes of
temperature, present work was carned on normal room temperature and hum~d~ty
Rats, l~ke other rodents have tremendous adaptab~l~ty to amb~ent cond~ttons The
we~ght of the ovary was found reduced In Intact mlce when subjected to h ~ g h
ambtent temperature (Narendranath and K~racofe, 1976)
Hypophysectom~sed hamsters shows dramatlc fall In ovarlan we~ght In the
1st week after surgery and thereafter, we~ghed approx~mately 113'~ of the normal
we~ght In the present work Intact and young female rats were used In different
~ntens~t~es of stress to assess solely the effect of stress In Intact rats
Delayed onset of puberty and reduced ovarlan slze was observed In
sem~starved (50% food restncted) an~mals L~ntern-Moore and Everltt (1978) has
proved that lnterrn~ttent starvat~on or sem~starvat~on causes stressful sltuatlons In
the present study, stress by starvat~on was not Intended, but ~t automat~cally got
Incorporated by the expenmental des~gn due to withdrawal of momentary food
depnvat~on dunng maternal separat~on and foot-shock stress In thls study ~t was
observed that soon after returning to home cages, from foot-shock session, rats d ~ d
not nrsh for food even when ~t was prov~ded Usually, on return to the cage they
engaged themselves rn explorat~on and then they consumed plenty of water This
post-stress act conf~rms acute dehydration and behavloural changes
Ovanes of female rat pups on DO welghed 0 77 mg In f~rst week the
Ovarian we~ght Increased by 162 33% and a week after the ovarlan welght
Increased 461 03% of blrth we~ght In~tlally, maternal depnvatlon stress d ~ d not
show slgnlflcant reduct~on in the ovarlan we~ght between control and stressed rat
Pups However, by end of 3'd week, the ovary of stressed rats were 8 15% lesser
than thelr counterparts In the control group (P < 0 05) From here onwards.
slgnlflcant differences exlsted In the ovarles of control and stressed groups Th~s IS
a clear ~nd~cat~on of cumulated effect of maternal deprivation The effect of stress
became more s~gn~flcant (P < 0 001) from D63 The cumulated effect of stress on
ovarles were so profound that after cessation of stress from D71 till D l00 ovarlan
we~ght could not come at par w~th control rats
Prlmord~al foll~cles have been described var~ously by d~fferent authors
(Pedersen and Peters, 1968, L~ntern-Moore et al, 1974, Gougeon and Cha~ny,
1987, Gaytan, 1997) In the present work a folllcle has been des~gnated as
primord~al, when ~t had oocyte surrounded by slngle layer of squamous type of
ep~thel~al cells In the later stages, the rlng of folllcular ep~thel~um was a mlxture of
flattened and cuboidal cells
As the age advances, the populat~on of the prlmordlal foll~cle decl~nes w~th a
slow pace, therefore, a negatlve correlat~on was found between age and number of
foll~cles Th~s substantiates the reports of Gougeon and Chalny (1987) that all small
follicles decrease with advanc~ng age Decltne In the number of primord~al foll~cle
co~nc~des wlth the Increase In the number of prlmary folllcles Thus, in control rats
the normal sequence of event are, decrease In early stages of the folllcles and
Increase In later stages of the follicles However, Increase In atresla was
Independent of any folllcular type but ~t was associated wlth Increase in age
The dlameter of the prlmordlal folllcles shows negligible difference between
the groups but at later stages, I e from D56 onwards the d~ameter of the prlmordlal
foll~cles dlffered between the groups It may be a sign of cumulated effect of
chronlc stress The effect of stress appeared more on the number of prlmord~al
foll~cle than the dlameter of the foll~cle Whlle d~ameter of these folllcles In rats of
the present study ranged between 20-34 pm whereas In human neonates
(Forabosco et al, 1991) ~t was reported to be 45 58 pm 5 13 21 Absolute volume
of prlmordlal foll~cles were not affected much due to stress
Removal of thymus at early postnatal llfe has caused reduction or complete
absence of primordial folllcles durlng ovarlan dysgenesls Nishlzuka and Sakakura
(1971) and previously Genther (1931) and Moawad et al (1965) reported that X-
lrradlatlon affects the growth of pnmordlal follicle In the present study extreme
measures like removal of any organ or cell dlstruction of an organ was avoided,
only changes of phys~olog~cal condltlons were employed to observe the effect of
stress on healthy llfe of an organlsm Both klnd of stress have resulted in
cumulated effect of chronlc stress However, whlle both klnd of stresses were
found effective but not permanently damaging or lethal
Primary foll~cles were recognlsed by the large size oocyte with clear nucleus
and atleast one nucleolus The oocyte was surrounded by two to many layers of
foll~cular cells (early and late pnmaly foll~cles) Different ways of classlfylng the
pnmary foll~cles have been put forward (Pedersen and Peters, 1968, L~ntern-Moore,
1974) In the present study, the folllcles were recognlsed by thelr surrounding
granulosa cell layers and not by number of cells
S~mklns (1932) stated that the number of prlmary folllcles are more or less
WnStant from blrth to maturity, then the number beglns to fall Slmllarly In the
present Study In control rats upto D49 the number of prlmary folllcles were w~thln a
small range, thereafter ~t decllned gradually at a slow rate Stress depressed the
POp~latlOn of pnmary folllcles and ~ ts effect pers~sted for more than four weeks after
withdrawal of stress When stress becomes chronlc and continues upto D100.
stress causes stgnlflcant volume reduction of the prlmary folllcles
Probably due to added granulosa cells, the prlmary folllcular mass
Increased, lnsp~te of stress In the present study the range of primary folllcular
d~ameter on 07 was 50-80 um whlch was slmllar to human neonatal ovary rn whlch
the mean dlameter was reported as 56 82 pm by Forabosco et al (1991) From the
foregoing statement ~t can be derlved that whether human or rat, soon after blrth
pnmary folllcular dlameter IS around 50 urn
The denslty of granulosa cells of prlmary folllcles bu~ldsup slowly from 2"d
PN week but after lorn week ~t does not Increase anymore Stress suppressed the
rate of Increase In the granulosa cells of the prlmary foll~cles The secondary
folllcles possess large oocyte wlth dlstlnct nucleus and nucleolus and surrounded
by granulosa cells whlch have Increased more than the granulosa cells of pnmary
folllcles (Shackell et al, 1996) The secondary folllcles could be observed In varlous
stages for havlng antra of vaned size and number Pedersen and Peters (1968)
described secondary folllcles on the bass of thelr number of granulosa cells, and
slze and number of antrum A follicle havlng one small antrum or mult~ple antra
were consldered as secondary folllcle On the 14 day PN though few foll~cles wlth
very small antrum was observed In the present study, this was consldered as
secondary folllcle, thus follicular counts were reported from D l 4
Dlscr~mlnation of types of folllcle was vividly done by Pedersen and Peters
(1968) But In vlew of many other reports, ~t can be lnfered that ovanan foll~cular
development may be species and straln dependent Present descnption holds valld
for ovanes of wlstar rats only
Stress affected the mean populatlon of secondary foll~cles from early stage
(014) to well advanced adulthood (D70) Such prolonged stress effect could not
show marked letup. 30 days after w~thdrawal of stress Besldes the populatlon, the
diameter of the secondary foll~cles showed some effect of stress but when the
range of the folllcular drarneters were presented metr~cally, then overlapping of the
dlameter of the secondary follicles was obviously observed Concomitantly, stress
s~gniflcantly affected the absolute volume of the secondary follicles after 17 days of
stress treatment Thls shows that during the penod of maternal deprlvatlon, stress
effect on absolute volume of secondary follicle IS not found but ~t IS also probable
that cumulated effect of maternal deprivation could manlfest only at the end of
preweanlng penod D21 Stress does not br~ng about marked changes In volume
dens~ty of granulosa cells upto 5m week but prolonged stress with Increased
intensity of foot-shock treatment slgnlficantly suppressed the granulosa cell denslty
of the secondary ovarlan follicles
A follicle was des~gnated as large follicle when ~t had a healthy prominent
oocyte havlng a clear zona pelluclda enveloped by corona radiata and suppolted by
C U ~ U ~ U S oophorus In a slngle large antrum Pedersen and Peters (1968) labelled
thew large follicle as Type 7 and 8 both havlng >600 foll~cular cells Whereas the
type 7 does not show a stalk of cumulus oophorus, the type 8 (preovulatory follicle)
holds the oocyte by a stalk of cumulus oophorus L~ntern-Moore et al (1974)
deflned large follicle as class F havlng >700 folllcular cells wlth all other features of
type 7 of Pedersen and Peters In the present work a follicle was considered as
large ~f it had features resembling Pedersen's type 7 and 8 and Llntern-Moore's
class F In the present study ~t was observed that few large folllcles were present
from D21 Kent (1972) have shown the presence of Graaflan foll~cles from D20 In
mlce
The population of large follicles In control rat Increases wlth age, but after
D42, ~t rema~ned wlthln a llmlted range Due to electrical footshock stress, as the
penod Increased the population of large foll~cles decreases and its effect pers~sted
even when the stress was not there In rats, foll~cles grow faster in earl~er l~ fe than
later penods, and more follrcles start to grow between 8-16 day than In 21 day ones
(Hage et al, 1978)
As the female pups become young adults, the dlameter of thelr large folllcles
Increased but the size vaned wldely Stress depressed ~t It IS poss~ble to flnd
progressive Increase In the absolute volume of large foll~cles whlch was severely
affected by foot-shock stress One can observe, that the thickness of membrana
granulosa of large follicles were thlnner than the secondary folllcles A remarkable
obse~ation of thls study IS that the large ovarlan folllcles of stressed rats showed
few indentations of varlous degree in membrana granulosa Consequent upon, the
nelghbounng theca interna also showed lnfoldlngs The volume denslty of
granulosa cells of the large folhcles are reduced by stress in the same way as in the
secondary follicles
Stress has negllglble effect on the antrum of large follicles It has been
found that due to stress there is sl~ght increase in the antral diameter This change
may be because of thinning of the granulosa layer
Hypophysectomy affects the large folllcles by markedly curtalllng the
folllcular maturation (Smith, 1930, Paesi, 1949), it decreases the percentage of
vesicular foll~cles (Lane and Greep, 1935), and also reduces the size of the large
follicles (Gougeon and Chainy, 1987), hypophysectomy may damage the large
folllcles causing atresla (Dufour et al, 1979) Dlameter of oocyte and dlameter of
follicles grows concomitantly till oocyte reaches its maximum growth, thereafter the
growth of the oocyte ceases but the folllcular growth continues (L~ntem-Moore et al,
1974) The present study substantiates the forego~ng statement, In add~t~on in the
present findings ~t was observed that the growth of the oocyte progressed through
the pnmary follicular stage then ~ t s growth remalned w~thln limited range in the
secondary follicle, and it stopped ~ t s growth before the follicle became matured
The volume of oocyte were more affected by stress during its proliferating
stage but not after the cessation of ~ t s growth Oocyte development IS not age-
dependent (Kramp et al, 1969) After birth no new oocyte are formed and the total
number of oocytes decreases as the rats grow older There IS slmllanty in the
growth of oocyte jn mlce and rat (Arendsen de Wolff-Exalto and Groen-Klevant,
1980) In mlce, the dlameter of oocytes was reported as ranging between 20-70
(Krarup et a!, 1969) In the present study, it was found that the dlameter
reached upto 65 05 pm
Oocyte plays an active role In organlzlng the granulosa whrch In turn IS
responsible for different~at~on of theca of the surrounding ovanan stroma (Knrgge
and Leathem, 1956) Oocyte d~ameter and foll~cular dlameter has a blphaslc
relatlonshlp They are positively and llnearly correlated unt~l the oocyte reaches ~ t s
rnaxlrnum growth (Knigge and Leathern, 1956, Llntern-Moore et al, 1974) Positlve
correlatlon has been observed between the oocyte and the follicles of rats growing
under stressed condltlons
Progression of the oocyte growth generally ceases at transition from Class D
to E (I e , secondary follrcular stage) but exceptionally rt may cease as early as
Class C/D (prlmary foll~cular stage) (Lintern-Moore et al, 1974) Oocyte growth
ceased at the end of secondary foll~cular stage In the present 0 b ~ e ~ a t l O n The
growrh rate of oocyte was not followed In the present study but reference can be
made (Gougeon and Cha~ny, 1987) that growth rate of the oocyte IS slower than
that of the follicular growth which in turn IS slower than the granulosa cell number
Present observat~ons lndlcate that cessation of growth of nucleus co~nc~des
w~th cessation of oocyte growth Similar results were reported by Lintern-Moore et
al (1974), In human ovary In which the oocyte nuclear d~ameter was reported as 26
pm Mereas In the rat ovary of present work, the oocyte nuclear diameter reached
~ t s maxlmum at 21 pm Positive correlatlon exists between the growth of oocyte
and growth of ~ t s nucleus, In humans (Llntem-Moore et al, 1974, Gougeon and
Chalny, 1987) In Gerbll (Guraya, 1985, Parshad et al, 1989, Saldapur and Kamath,
1993)
The findlng of the present study has shown varlous degree of effect of
stress on the ovarlan follicles lncludlng the oocyte can be correlated w~th the
observations reported from thls laboratory (Mukerjee, 1987) whlch states that total
PoPulatlon of gonadotrophs and cytoplasmic volume of gonadotroph cells were
slgnlflcantly decreased due to chronlc foot-shock stress
In the present study corpus luteum was seen from D56, stress delayed
formation of corpus luteum for one week Later appearance of corpus luteum may
be due to delayed onset of flrst ovulatlon Delay In the ovulatlon may be a result of
lsolatlon of the female rats and absolute non-contact w~th the male rats Female
rats used In the present study d ~ d not have chance of stlmulatlon even by olfact~on
Stress affects numerical denslty of small and large luteal cells, consequently
affecting the absolute volume of the whole corpus luteum Slmllarly, as the mean
volume of each luteal cells and the volume denslty of thelr components (cytoplasm
and nucleus) have shown moderate to severe effect of chronlc stress Comparable
effects of llght and dark cycle (Pomerat, 1942) and cold-exposure (Re~ter, 1968)
have shown reduction In the slze and amount of luteal tlssue
Removal of thymus have been reported to cause absence of corpus luteum
In mlce (Nlshlzuka and Sakakura, 1971) In the present work, stress affected the
thymus and ~ t s funct~onal components are presumably decreases the LH level
Concomitantly, stress affected the ovarlan components lncludlng corpus luteum ~n
stressed rats
One of the commonly found feature of an ovary IS presence of atretlc foll~cle
Engle (1931) reported, In mlce atretlc folllcles are found about 2oL day and this
number IS falrly constant untll puberty In the rats of the present work, earllest s~gns
Of atreSla was noted on 028 Kn~gge and Leathem (1956) dtd not observe slgn of
atresia in preantral follicle of hamster ovary upto D28
Throughout the reproductive l~ fe atres~a continues so that 99 9% of all the
follrcles become atretic and only 0 1% eventually ovulate (Hlrshfleld and M~dgley,
1978) Many follicles grow durlng each cycle and reach a large slze, but only a
small percentage attaln preovulatory stage and undergo ovulation (Myers et al,
1936, Willtams, 1956, Loeb, 191 1)
Thls work fully substantrates the flnding w~th the present observation
show~ng progressive atresia from D28 to 0100 PN In the ovarles of Immature
anlmals, atresla of preantral follicle beglns several days prlor to in~t~al ovulatton and
then proceeds regularly with the constant number (Kn~gge and Leathem, 1956)
Several authors have attr~buted (Greep et al, 1942, Kn~gge and Leathem, 1956) LH
to be responstble for the settlng In atresla of small or premature folllcles Thls IS
one of the cause of Increased atresia of small follicles durlng ovulation Similarly, in
gulneapig large atret~c folllcles were found dur~ng ovulatton (Myers et al, 1936) As
ment~oned earl~er atres~a IS a regular process In mouse, Byskov (1974) have
descnbed that ~t takes 3-4 days for a healthy folllcle to reach an advanced stage of
atresia In rats, large foll~cles become atretic 3'd day of the cycle (Bollng et al,
1941) The reason for atresla of medlum and large slzed folltcles may be many that
Includes all klnds of stresses whlch Interrupts the occurrence of regular estrus cycle
which acts via hypothalamo-hypophyseal-adrenowrt~co-gonad axis
It has been reported that many vesicular foll~cles turned atrehc as a result of
sem~starvat~on, the condltlon which resembles the hypophysectomy In rats (Llntem-
Moore and Ever~tt, 1978) After four days of hypophysectomy all the pnmordlal
folllcles have been reported to have undergone atres~a (Sm~th, 1930) Although,
Byskov's (1974) classlflcatlon of atres~a was not taken Into conslderatlon, atretlc
foll~cles were ~dent~fled on the bas6 of crlterla described by other authors (Brand
and DeJong, 1973, Osman, 1985)
In the present work dunng the preweanlng per~od when maternal deprlvat~on
stress was executed, lnlt~ally no atretlc foll~cles were observed However, In the
stressed rats atret~c folllcles were found on D21 Two weeks after contlnued
execution of foot-shock stress, the atretlc process was enhanced rapldly Two
weeks after withdrawal of stress, rats have shown decreased number of atretlc
folllcles Thls lndlcate that atretlc process has reduced to some extent
The organlsat~on of stromal cells takes place by the end of flrst week PN
The oocyte In large number are found w ~ t h ~ n the elongated or flattened stromal
cells Though denslty of stromal cells 1s qulte less on D7, ~t rapldly contlnued to
grow denser and tn a perrod of 100 days, the absolute volume of stroma Increases
by 26 57 tlmes The present study shows reduction In the absolute volume of
stroma In the stressed group of rats It needs to be underl~ned that they showed
hypertrophy of the lnterstlt~al cells In the ovary of stressed group of rats
The ~nterstttral tissue forms a consprcuous component of the ovary as early
as 14 day of age in rats (Dawson and McCabe, 1951; Rennel, 1951). The present
study indicates that in control rats atretic process must have started after 3 week
PN age, hence the interstit~al glands were observed from D28 onwards
Besides the effect of stress on the ovary which caused hypertrophy of
interstitial gland, other causes for hypertrophy were light deprivation and cold
exposure (Reiter, 1968), due to thyrnectomy (Nishizuka and Sakakura, 1971;
Michael et al, 1980) and FSH treatment Gonzalez-Moran and Mantilla, 1998).