prolonged exposure to stress

Plasma

testosterone

levels and

_{\g=D\5-3\g=b\-hydroxysteroid}

dehydrogenase

activity

in the

testis of

the

rat

following

prolonged

exposure

to stress

Irina

_Pollard,

J.

R.

Bassett and

Jean M. P.

Joss

School

_ofBiological

_Sciences,

_{Macquarie University,}

North

_Ryde,

NewSouth

Wales,

Australia 2113

Summary. Exposure

of male CSF rats to a

signalled,

unpredictable

60-day

stress

regimen

induced a

significant

elevation in

circulating

testosterone levels at

_Days

1

and 60which was abolished

by

castration. Small

morphological

changes

were seen in the

_Leydig

cells in the

_early

days

of

_stressing.

A

_significant

increase in

_3\g=b\-HSD

activity

was seen

_by

5

_days

of stress,

_indicating

increased

_{steroidogenic activity.}

Introduction

Exposure

toa stress

_procedure

may elicit a

multiplicity

of hormonal_responses

which,

through

their metabolic

effects,

are

interdependent

and can lead to disturbed

_{physiological}

_changes.

Pollard,

Bassett & Cairncross

₍₁₉₇₆₎

describedanultrastructural

study

of the

adenohypophysis

in stressed rats, in which the cellular

activity

of the

_{corticotrophs}

correlated wellwith the circu¬

lating

levels of corticosterone and the

_gonadotrophs

showed marked

_{morphological changes.}

Both the

_{follicle-stimulating}

hormone

_(FSH)

and the

_luteinizing

hormone

_{(LH) gonadotrophs}

showed intensesecretory

_{activity during}

theinitial 10

days

ofstress.

By

20

days,

_{gonadotrophic}

activity

returnedtoacontrol

level,

but therewasthenaninhibition of

activity

at60

_days.

Plasma testosterone levels have been

_reported

to

_change

_following

exposure to stressful

situations,

but the data appear to be

_{contradictory}

and therefore difficult to

_interpret.

For

example,

during

intense

_physical

exercise

_plasma

testosterone levels have been

reported

to

increase

_{(Dessypris, Kuoppasalmi}

&

_Adlercreutz,

_1976),

decrease

_(Sutton,

_Coleman,

_Casey

&

Lazarus,

1973),

orremain

unchanged

(Lamb, 1975).

It

is,

however,

accepted

that Stressorsdo elicit

_changes

in testicular

_secretion,

and the type of stress used could have caused different stress-induced

_changes

intestosteroneconcentrations.

In this

_report

the effects of

prolonged

stress on testicular secretion oftestosterone were examined and correlatedwithultrastructural

changes

inthe

_Leydig

cells ofrats.

Materials and Methods

Animals and

experiments

The male CSFratswere87-93

days

oldwhenused. The animalswerehoused in groups of 3 under conditions ofconstant temperature and

_humidity

(21

±

0-5°C,

46%

_humidity)

and with

12 h

_{light (20:00-08:00}

_h)/24

h,

_beginning

at least 14

_days

before the

_experiment

and con¬

tinuing

throughout.

Foodandwaterwere

always

available.

0022-4251/80/030101 -07S02.00/0

The

_experimental

animals used for thetestosteronemeasurements andultrastructural studies were stressed for

1, 5,

10, 20,40

and 60 consecutive

days

andratsfor

_study

of the

_activity

of

3ß-hydroxysteroid

dehydrogenase

(3ß-HSD)

inthe

_Leydig

cellswerestressed for

1, 3,

5, 10, 20,

40 and 60

_days.

The stress

_procedure

used was that describedin detail

by

Bassett,

Cairncross &

King (1973).

Animals were

placed

in automated one-way avoidance boxes

_(Lafayette

Model No.

_85200).

Anescape

platform

was availabletothe animal viaanautomated

moving

partition.

A

_{light-conditioned}

stimulus of 2 W was located onthe wall of the

grid

chamber

opposite

the escape

platform.

The unconditioned stimulus was delivered

by

a

generator-scrambler

through

the

grids

as a2

mA,

50

pulses/sec

squarewave.Eachratwas

placed

onthe

platform

atthestart

ofthe treatment session. On eachtrial the conditionedstimulus started4 secbeforethe animal was

pushed

by

the movable

partition

from the

platform

onto the

_grid

and the unconditioned stimulus

_began.

The movable

_partition

wasthen

immediately

retracted and the animalwas _able

to

_jump

from the

_grid

tothe

re-exposed

_platform

withaminimum

latency

_{of 0- 3}sec.Theuncon¬ ditioned stimulus was

stopped

by

the return of the animal tothe

_platform.

This

_irregular

_sig¬

nalled foot-shock

_procedure

is

_proposed

as

having

a

large psychological

component

(Bassett

et

al, 1973).

Each

_day

the rats were

subjected

to 7

_randomly

timed shock treatments. Immedi¬

ately

after the last

session,

the animals were killed

by

cervical dislocation and

exsanguinated.

The blood

_samples

were collected and the

plasma

wasfrozen. All

samples

were collected in the

morning

between 9:00 and 12:00h inordertominimize diurnal variations.

This treatment

_procedure

was

repeated

with castrated animals. Rats were castrated under ether anaesthesia and after 15

_days

onegroupofcastrated animalswas stressedfor

1, 5, 10, 20,

40or60

days

andtheothergroupwasleft unstressed

(controls).

The controlswerekilled atthe

sametime

periods.

Therewere6 stressedand3 control

animals/time period.

Testosterone assay

Testosterone was

assayed by

the

radioimmunoassay

kit

supplied by

New

England

Nuclear

(Biomedicai

_{Assay Laboratories).}

Testosterone was extracted from

plasma

using

dichloro-methane;

no

Chromatographie

separation

was carried out. The freeze-dried antiserum is pre¬

pared

in rabbits

_against

testosterone-3-oxime-bovine serum albumin.

_{Cross-reactivity}

at 50%

displacement only

occurred to any

appreciable

extent with

_{dihydrotestosterone (100%).}

Unbound

_{[l,2-3H(N)]testosterone}

was removed fromthe incubation mixture

by using

dextran-coated charcoal. The

_sensitivity

of the assay was 0-01

ng/ml

and the inter- and

intra-assay

variationwere_{each <10%.}

Electron

microscopy

Thetestesfrom 5 stressed and 2controlanimalsfrom eachgroupwere

rapidly

removed and

placed

in 4%

_{glutaraldehyde}

in 0-1 M-sodium

_cacodylate

bufferfor 2 h at 5°C.Thetissueswere then

_post-fixed

in 2% osmium tetroxide for 1-5

_h,

_dehydrated,

and embedded in Araldite

(CIBA).

Silver sections werethen stained

by

the

triple

stain method modified fromSoloff

(1973).

The

_sections,

whichwere mounted on copper

_grids,

were treatedwith a0-9%

KMn04

_solution buffered at

pH

6-5 with

_phosphates

for 2 min.

_They

were then treated with

uranyl

acetate

saturated in 50% ethanol for 3

min,

followed

by

0-2% lead citrate solution for 3 min. This

method of

_staining

increased electron

_density

of the membrane systems in

_particular,

_which

greatly improved

cellular definition. The

_grids

were examined in a JEOL 100CS electron

microscope.

Assay

for 3ß-HSD

Immediately

after the laststresssessionratsfrom thecontrol andstressedgroupswerekilled and thetestes were

rapidly

removed and frozenin

liquid nitrogen.

Testicular

_3ß-HSD

was determined

by

the method ofPearse

(1972).

The 3 substrates used were

pregnenolone, dehydroepiandrosterone (DHA)

or

epiandrosterone

because

3ß-HSD

con¬

verts

_pregnenolone

to progesterone

(a

more _{distant precursor oftestosterone}

steroidogenesis)

and DHA and

_{epiandrosterone}

to androstenedione and androstanedione

_respectively

(more

immediately

involved intestosterone

metabolism).

In the_{presence of}NADas acofactorthe blue

reduction

_product

of

tetrazolium, formazan,

marked the location and

_intensity

of

enzymic

activity.

The

intensity

of this blue colour was rated onan

_arbitrary

scale from0

_(absent)

to 5

(intense) by

visual examination oftheslides.

Results

Testicular

_weight

No

_{significant changes}

in wet

_weights

of the testes were observed for stressed and control

rats overthe entire

60-day period.

The mean ± s.e.m. wet

_weight

values were 1-68± 0-06 and

1-70 ± 0-08gfor the

right

testesof stressed and controlrats

respectively.

Plasmatestosterone

The results are

given

in Table 1. A one-way

analysis

of variance showed no

significant

differencebetween the control_groups

_(intact

or

castrated)

overtheduration ofthe

experiment

(F

=

0-37,

d.f. 5,

17;

>

0-05)

so the control groups were

pooled.

The

plasma

testosterone

levels for the stressedratsshowed a

biphasic

pattern,

being

significantly

high

on

Days

1 and 60.

Circulating

testosteronewasverylow inthe castrated controlratsand values in thestressedand castratedanimalswerenot

significantly

different

throughout

the

experiment (Table

1).

Table 1. The effects of

_prolonged

exposure to stress on mean + s.e.m. testosterone levels in intact and

castratedrats

_(no.

in

_parentheses)

Plasmatestosteronelevels(ng/ml)

Daysofstress Intact Castrated

Pooled control 3 1 *4 5 4 10 3 20 3 40 3 7±0 8±0 2±0 9±0 4±0 4±0 1(18) 0-08±0-01(18) 5(6) 0-09 ± 0-01 (6) 3(6) 0-10±0-01(6) 3(6) 011 ± 0-005(6) 1_{(6) 0-07 ± 0-005(6)} 4(6) 0-10_±0-01(6) 60 *4-4 ± 0-3(6) 0-07 ± 0-005(6) *

Significantly different from the _pooled control value, <0 02(unpaired/_tests).

Leydig

cellultrastructure

The fine structureof the

_Leydig

cells of control animalswas similartothatdescribed in the literature

_(Lacy

&

Pettitt,

1969).

In

general,

stressdidnotcausegreat

cytological

alterationsand no

pathological signs

wereobserved.

However,

small ultrastructural

changes

wereobserved after 5

_days

ofstress.

By Day

5the

Leydig

cells showedanincrease ofboth smoothand

rough

endo¬

plasmic

reticulum as well as free ribosomes and this was more marked after 10

days

ofstress

whentherewasalso a

depletion

ofthe

lipid

droplets,

bothin number_presentand size oftheindi¬ vidual

_droplets,

many

_being

_collapsed

and crenated in appearance.

By

10

_days

_manyof the

mito-chondria had

changed

froma

spherical

oroval

shape

with

closely

packed

tubular

cristae,

as seen in the control

animals,

to structureswhich had

_enlarged

and showed

cavities,

broken cristaeand

dense inclusions.

After 20

_days

ofstress mostof the above

_changes

werestillobservablebuttoa_{lesserextent.}

By

40 and 60

days

of_stress,the

_{lipid droplets}

had been

_replenished

andthemitochondria and other cellular

organdíes

werenormal.

Activity

of3ß-HSD

The

_{3ß-HSD activity appeared}

to be

_strictly

limited tothe

Leydig

cells,

and there was no

trace of

_activity

in the tubular cells. Formazan

_deposits

of various intensitieswere found in all sections incubated with substrate whilstthesections

_lacking

substratehadno_formazan

deposits

(PI.

1,

Figs 1-4).

As shown inTable2 therewas a

significant

increasein the

density

of formazan

deposits

withDHA and

_{epiandrosterone by}

3

_days

ofstress and

_by

5

_days

all three substrates

gave

_{significantly}

elevated formazan

_deposits.

The enzyme

activity

towards DHA remained elevated for 10

_days

ofstress before

_falling

tocontrol levels. When

_pregnenolone

and

_epiandro¬

steronewereusedas

substrates,

formazan

deposits

felltothecontrol level

by

10

days.

Table 2. Mean ± s.e.m.

_{activity (scored}

1-5, see

_'Methods')

of

5-3ß

hydroxysteroid dehydrogenase

(3ß-HSD)

in thetestesofrats

_exposed

to stress

₍₆

rats/group,

3rats/control

_group)

Substrate

Daysofstress

Pregnenolone Dehydroepiandrosterone

Stress Control Stress Control

Epiandrosterone Stress Control 1 3 5 10 20 40 60 1-0±0-4 2-0±0-4 2-3+0-2* 1-8+_0-8 0-6±0-2 0-6+_0-2 0-8+_0-4 3±0 7±0 •7+0 •3±0 •3±0 •7±0 •3±0 2-4±0-2 2-6±0-2* 4-0±0-3* 3-0±0-3* 1-4±0-4 1-8±0-2 2-0+_0-4 1-7±0-3 1-3±0-3 2-3±0-3 1-3±0-3 1-3±0-3 l-7±0-3 2-3+_0-7 •6±0 •6+0 •8±0 •4±0 •6+0 •0±0 2±0 •3±0-7 ·0±0·6 ·0±0·6 •7±0-3 •3±0-3 •3+_0-3 •3±0-7 *

Valuessignificantlydifferent from therespectivecontrolvalue, <0 05(unpairedt_tests).

Discussion

The stress-induced

changes

in testicular

function,

as reflected in

plasma

testosterone

levels,

correlate well with

_{morphological change}

in the

_Leydig

cells. The rises in

_plasma

testosterone

evident at

_Days

1 and 60were

completely

abolished in castrated animals

subjected

to asimilar

regimen,

demonstrating

that thetestosterone concentrationsin_{the stressed animals} weredueto

testosteronesecreted

_by

the testis and not

_by

the adrenal

gland.

The rangeof

circulating

levels of

testosterone for both the intact and castrated animals is in

good

agreement with those of other

investigators (Verjans,

van der Molen &

Eik-Nes,

1975;

Pujol, Bayard,

Louvet &

Boulard,

1976).

The described ultrastructural

changes

in the

_Leydig

cells are all indicative of

augmented

secretory

activity.

The

_precise

role ofthe

_{lipid droplets}

in

_{steroidogenesis}

is stillnotclear but it is

thought

that the

_lipid

droplets

inthe

_Leydig

cells functionasstorage

depots

ofprecursorsusedin the

_synthesis

of

androgens (Connell,

1976).

Other

investigators

haveobserved that the

_droplets

PLATE 1

Micrographsoftestesfromrats

_subjected

to3daysexposuretotheStressor.

Fig.

1. Control section

_lacking

substrate (score 0).

Figs 2-4.

_Arbitrary

values of_enzyme

_activity

with

_{dehydroepiandrosterone}

as _precursor of scores2,4and 5

_{respectively,}

98.

been shown

(Pollard

et

al,

1976)

that in animals stressed for 10

days

there was an extensive increased

activity

in the LH

_{gonadotrophs, presumed}

indicative of

_pituitary

secretion in the absence ofan available _assayfor

plasma

_{gonadotrophins,}

but

by

60

days

the

activity

_exhibited was _{below that of the controls. The extension of the}

rough

_endoplasmic

_reticulum

together

_with free ribosomes in the

_Leydig

cells in the present

study

is considered to be a reflection ofthe increased

_synthesis

ofsmooth

_endoplasmic

reticulum

_following

demandsofincreased

_androgen

synthesis.

The observed mitochondrial

changes

at 10

days

mayalso bea responseto

high

circu¬

lating

levels ofLH. Gallon & Dufaure

(1975)

described similar alterationstothemitochondrial ultrastructure in _rat luteal cells incubated with LH. Mitochondrial conversion of cholesterol to

pregnenolone

appearstobe a

rate-limiting

_step in the steroid

synthesis

ofthe_{testis and}

gonado¬

trophins

might

stimulate mitochondrial

_activity

for

_{pregnenolone production}

(Van

de

Vusse,

Kalkman,

Van Winsen & van der

Molen,

1975).

The reason for the increased

plasma

testos¬ terone concentrations at

_Day

60 ofstress is uncertain since no mitochondrial alterations were observedatthis time

(present study)

and there were reduced ultrastructural

changes

in the LH

gonadotrophs (Pollard

et

al,

1976).

In the

_{psychologically}

conditioned animal the hormonal

_profile

_may

_only

_represent

alterations in energy metabolism. Plasma testosteroneis

_{significantly}

increased after strenuous

running

exercise and the effect is more

pronounced

in the fit runner than the non-fit runner

(Kuoppasalmi,

Naveri, Rehunen,

Härkönen &

_Adlercreutz,

_1976).

Sutton et al

₍₁₉₇₃₎

found that

physical

exerciseraised serum

androgens

and that this risewas

independent

ofserum LH

values,

perhaps

because of reduced

_hepatic

clearance of

androgens.

A decreased clearance of

testosterone could be

_responsible

forthe elevated

plasma

testosterone in the ratsofthe _present

study

which had habituatedtothe

_{psychological}

Stressor

by

60

_{days (Pollard}

et

al,

1976).

Thisresearchwas

supported

by

the Australian Research Grants

Committee,

GrantNo.

Dl-73/15017.

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