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).