(Received 12th June 1968)

PLASMA LEVELS OF TESTOSTERONE

IN

MALE

RABBITS FOLLOWING COPULATION

GARY C. HALTMEYER and KRISTEN B. EIK-NES

Department

of

Physiology,

Division

_of

_Physiology

and

_Biochemistry

_of

_{Reproduction,}

University of

Southern

_California,

Los

_{Angeles, California}

(Received

12th

_June

₁₉₆₈₎

Summary.

Normal,

male rabbitswere either allowed to

_copulate

with

receptive

females,

or were

injected

with human chorionic

_{gonadotrophin}

(hcg),

or with

adrenocorticotrophic

hormone

(acth).

Copulation

and hcg

injection

produced

significant

increases in

plasma

testosterone

while acth administration failed to do so. Rabbits treated with either

chloropromazine

or

fluoxymestrone

before

_copulation

did _not increase

plasma

levels oftestosterone

_following

coitus.

INTRODUCTION

It is well established that the rabbit ovulâtes _{in response} to

_mating

and a

marked ovarian release of

_{20a-hydroxypregnen-4-en-3-one}

will occur

shortly

aftercoitus in this

_species

_(Hilliard,

Archibald &

_Sawyer,

_1963).

This

_change

in

progestin

secretion

_following

_{mating corresponds}

with the increase of

_gonado¬

trophin

in

_peripheral

blood

_{(Hilliard, Hay}

ward &

_{Sawyer, 1964),}

and the

release of interstitial cell

_stimulating

hormone

_(icsh)

and

_prolactin

from the

pituitary

gland

(Desjardins,

Kirton &

_Hafs,

_1967).

_Moreover,

_Taleisnik,

Caligaris

&Astrada

₍₁₉₆₆₎

notedadecrease in

pituitary

contentoficshand an

increase in

_plasma

icsh infemale rats

_{following copulation.}

The level ofascor¬

bic acid in the_ovaryof these animalswas lower than in controlrats.

The_{hormonal response of the male}

_gonad

to

copulation

is lessovert and has

received little attention.

_Copulation

will increase the blood levels oficsh and

decrease the

_pituitary

concentration ofthehormone inthe male rat

_(Taleisnik

et

_al.,

_1966).

The secretion of

_androgenic

steroids into the

_spermatic

venous

blood of anaesthetized rabbits appearstoincrease

_shortly

aftercoitus

_(Endröczi,

1962)

and this increase isnotfoundin malerabbits with

_hypothalamic

lesions. Since testosteroneis the

_major

_androgen

secreted

_by

the male

_gonad,

it was

deemed

_important

to

study

fluctuations of thishormone in the

_peripheral

blood of the rabbit

_following

coitus.

METHODS

Adult male rabbits of the New Zealand strain were used. The animals were

maintained in individual wire _cages,

_given

unlimited food and water and

housed outdoors.

_Experiments

were conducted

only during

the winter season.

Gary

Haltmeyer

Twenty

millilitres ofbloodwerecollected

by

heart_puncturefromeach of the

animals before

_{experimentation}

toestablish their

resting

levels of

plasma

testo¬

sterone.Ten animalswere

assigned

toeach of the

following experimental

condi¬ tions:

(a)

no _treatment

(resting

level

control)

_;

(b) copulation

with a

receptive

female

rabbit;

(c) injection

of500 i.u. ofhcg

intravenously;

(d) injection

of5

_i.u./kg

ofacth

intravenously;

(e) copulation

with a

receptive

female

following

subcutaneous

injections

of15

_µg/kg

of

_{fluoxymestrone (9a-fluoro-ll/?-hydroxy-17a-methyl}

_{testosterone)}

at

18,

12 and 6 hrbefore collection of bloodfor measurementof

_resting

level of

testosterone;

(f) copulation

with a

receptive

female

following

intramuscular

injection

of

3-5

_mg/kg

of

_{chloropromazine (CPZ)}

1 hrbefore collection of bloodforestima¬ tion of

_resting

level oftestosterone.

A

_sample

of20ml ofbloodwas collected

by

heart _puncturefromall animals 45 min after

_copulation

or treatment with hcg or acth.

Fluoxymestrone

and

CPZ were

given

in an

_attempt

to block the

_changes

of testicular

_activity

induced

_by

_copulation.

In order to determine ifthese

drugs

had inhibited the

ability

of the testis to

_produce

_{testosterone,} the

_{fluoxymestrone}

and CPZ

animals were

given

a

subsequent

intravenous

injection

of 500 i.u. hcg and blood was taken

(by

heart

puncture)

for testosterone estimation 45 min later. The blood

_samples

were collected in

heparinized

vacutainers,

immediately

placed

on

ice,

centrifuged,

and the

plasma

removed and

assayed

for free testosterone

using

the method

published by

our

laboratory (Brownie,

van der

Molen,

Nishizawa &

_Eik-Nes,

_1964).

The

_specificity,

_{accuracy and}

_precision

ofthis methodhave been demonstrated

_(Brownie

et

_{al., 1964;}

Resko &

_Eik-Nes,

1966).

RESULTS

Since the

_resting

level of

_plasma

testosterone was determined in each animal

before the

_{experiment began,}

each rabbit served as its own control and a

statistical

_design

of within

_{subject analysis}

of variance was

employed.

Use of

this

_type

ofcontrol

_experiment

was _necessarybecause ofa rather

large

seasonal and individual variation in

_plasma

testosterone which exists in the rabbit.

Since both the main effects and their interactions were

significantly

different

from the controls

_{(Table 1),}

an

analysis

of

simple

effects was

computed.

This

analysis

indicatedno differences between the

resting

levels of

plasma

testoster¬

one in each ofthe

experimental

_groups, the meanlevel

being

37-6

ng/10

ml of

plasma.

Both

_{copulation (F}

=

6-88)

and _hcg administration

(F

=

41-43)

produced significant

increases in

_plasma

testosterone

_(mean

levels of 77-3 and

165-4 ng

testosterone/10

ml

_{plasma, respectively).}

Administration of acth

(Text-fig. 1)

failed to

_produce

an increase in

circulating

_testosterone and no

stimulatory

effect of

_copulation

could be found in animals treated either with

fluoxymestrone

or with CPZ before coitus

(Text-fig.

1).

Since these animals

Testosterone

_response

to sexual stimulation 275

hcg

(Text-fig.

1),

the lack of elevation of

plasma

testosterone

following

coitus is

probably

not due to inactivation of testicular enzymes

producing

this steroid

hormone.

Table 1

analysis of variance for plasma testosterone in male

rabbits following copulation

Sources_ofvariation Between_subjects

(treatment)

Subjectswithin_group Withinsubject A_(time) AB

AX_subjectwithingroup

SS 217,436-7 119,740-9 97,695-8 135,042-3 10,524-0 54,436-1 70,082-1 df 59 5 54 60 1 5 54 MS 23,948-2 1,809-2 10,524-0 10,887-2 1,297-8 13-24* 8-11* 8-39* *P<0-01. 2IOr-180 150 120 90 60 30 0 -30 -60

1

RLcontrol Cop hcg acth CPZ _Fluoxy and and

Cop Cop

Treatment

Text-fig. 1. Mean_{% change}from _restinglevel of_plasmatestosteronein malerabbits.

RL control:restinglevel 45minafter initial collection of blood.Cop:45min after_copula¬ tion,hcg:45 min after intravenoushcg. acth:45 minafter intravenousacth.CPZ and

Cop:_{rabbits treated withchloropromazine}andthen_permittedto_{copulate(shaded}area

indicates_responseto_{subsequent injection}of_{hcg). Fluoxy}and_Cop:rabbits treated with

fluoxymestrone and then permitted to_{copulate (shaded} areaindicatessubsequentre¬

sponsetointravenous_hcg).

DISCUSSION

The male rabbit shows a marked increase in

plasma

testosterone

following

Gary

Haltmeyer

obtained

_by

Endröczi

₍₁₉₆₂₎

who determined

_androgens

in

_spermatic

venous

blood of anaesthetized rabbits and to recentdata

_{by Saginor}

& Horton

_(1968).

The _responseof the male rabbit to sexual stimulation_appears,

therefore,

to be

similar to that of the

_female,

i.e.

_spontaneous

release of

_pituitary

icsh with

subsequent

stimulation of secretion of

_gonadal

steroids

_(Hilliard

et

al.,

_1963).

Administration of CPZ or

fluoxymestrone

before

copulation

blocked the

increase of

_plasma

testosterone associated with this

physiological

event. The

primary

effect of CPZ is

_thought

to be on the central nervous _system with no

appreciable

direct effect upon a tissue like the testis. This is

supported by

our

observation that animals treated with this

_drug

still show a normal

plasma

testosterone _response

_following

administration ofhcg.

Fluoxymestrone

would

be

_expected

toaffect the

_{hypothalamic-pituitary}

feedback

_system.

Prior treat¬

ment with

_{fluoxymestrone}

blocked the response to

copulation

and tended to

decrease the

_subsequent

effect ofhcg on

circulating

testosterone

(Text-fig.

1

_).

Fluoxymestrone

may thus have some direct effect _upon the testis aswell as its

expected

effecton the

hypothalamic-pituitary

feedback mechanism. It

should,

however,

be remembered that

_{fluoxymestrone}

was administered _every 6th hr

during

the

_day

before hcg

injection.

Treatment with

fluoxymestrone

for this

period

of time could have resulted in a

gradual

decrease of

pituitary

icsh

secretion

_and,

as a_{consequence of this}

change

in

pituitary activity,

the

capacity

of the testis to

_produce

and secrete testosterone

following

administration of

exogenous

gonadotrophin

may have also decreased

(Eik-Nes, 1964).

Whatever

the solution to these

_problems,

our data indicate that the central nervous

system

is involved in

augmenting

the secretion of testosterone in the male

rabbit

_{following copulation.}

Removal oftwo20-ml

_samples

of blood within45

min,

inananimal

weighing

about 3

_kg,

must be classified asa 'stressful' condition. Still this treatment did not lead to

augmented

levels of

plasma

testosterone

_(Text-fig.

1

_).

If

_anything,

these levels tended to decrease

_slightly.

This observation is in

keeping

with

previous findings

that the level oftestosterone in

spermatic

vein blood of the

dog (Eik-Nes,

1962)

and of the rat

(Bardin

&

Peterson,

1967)

will decrease

during

stressful conditions. Such conditions are known to increase

_pituitary

secretion of acth. This hormone _appears,

however,

to have no measurable

effectonthe cells ofthetestis

secreting

testosterone

(Text-fig.

1

).

In

spite

of the fact that icsh and acth show many similarities with

regard

to mechanism of action

_(Eik-Nes,

_{1964), they}

are most

specific

in

_selecting

the _{proper target}

organ

(Eik-Nes,

1962).

At

_present

there is no

explanation

for the association of

copulation

with an

increased secretion oftestosteronein the male rabbit. This animal is known to

copulate

very

rapidly

when

placed

witha

receptive

female and alsoto

_copulate

repeatedly.

Some workers consider testosterone tobe the hormone

influencing

the sex drive. The initial

ejaculation

is

probably completed

before the rise in

plasma

testosterone is

established,

though

time

experiments

with

regard

tothis

increase must still be done. Testosterone can affect _sperm maturation and is

present

in the

_epididymal

tissue of the rabbit

_(Frankel

&

_Eik-Nes,

_1968).

The

post-coital

increase in testosterone secretion could thus serve the purpose of

Testosterone

_response

to sexual stimulation 277 Macmillan & Hafs

₍₁₉₆₇₎

found that sexual

_preparation

before

_ejaculation

greatly

increased the _sperm countin the first and second

_ejaculate

_through

its effect _{upon the}

_{epididymides.}

Testosterone secretion _{in response} to sexual stimulation could also involve mechanisms associated with_sperm

_{capacitation.}

Finally,

the observed /reflex secretion' of testosterone

_following

coitus could reflecta

similarity

in the

design

ofthe

hypothalamic-pituitary

axis ofthe male

and the female

_rabbit,

and the

_augmentation

in testosterone secretion could serve noknown

biological

function. If_so, this reflexmechanism_maybe _present

in animals other than the rabbit and the rat

_(Taleisnik

et

_al.,

₁₉₆₆₎

since

preliminary

data from our

laboratory

indicate that the level of testosterone

mayincrease in the

plasma

of normal men

following

coitus. ACKNOWLEDGMENTS

This work was

supported

in _part

by

research _grants AM-06651 and TOI CA 5000 from the U.S. Public Health

_{Service, Bethesda,}

_Maryland.

REFERENCES

Bardin,C. W. &_Peterson,R. E.₍₁₉₆₇₎Studies ofandrogen productionbytherat:testosteroneand androstenedionecontentof blood._{Endocrinology,}80,38.

Brownie,A.C,van der_Molen,H._J.,Nishizawa,E. E. & _Eik-Nes, . .₍₁₉₆₄₎ Determination of

testosteronein human_{peripheral plasma using gas-liquidchromatography}with electroncapture

detection._J.clin. Endocr.24, 1091.

Desjardins, C,Kirton, K. T. & Hafs, H.D. _{(1967) Pituitary}levels of_lh, fshandprolactinafter

coitus inrabbits. _(Abstract).Fedn Proc. Fedn Am. Socs_exp.Biol.26,534.

Eik-Nes,K.B. (1962) Secretion oftestosteronein anesthetized_{dogs. Endocrinology, 71,} 101.

Eik-Nes, K. B. ₍₁₉₆₄₎ Effects ofgonadotrophins onsecretion of steroids bythe _ovaryand the testis.

Physiol.Rev.44,609.

Endröczi,E._{(1962) Copulation}induced testicular secretioninthe male rabbit._ExcerptaMed.Internat.

CongressSeriesNo.47,620.

Frankel,A. I.&Eik-Nes,K. B._{(1968)Steroidogenesis}in vitroof the_epididymisof the rabbit.(Abstract).

Fedn Proc.Fedn Am.Socsexp.Biol.27,624.

Hilliard,J., Archibald,D. &Sawyer, C.H. _{(1963) Gonadotropic}activation of_{preovulatorysyn¬} thesisand release of_progestininthe rabbit._{Endocrinology,}72,59.

Hilliard,J.,Hayward,J.N. & Sawyer, C. H. ₍₁₉₆₄₎ Postcoital_patterns of secretion of_pituitary

gonadotropinand ovarian_progestinintherabbit.Endocrinology,75,957.

Macmillan, . L. & Hafs, H. D. ₍₁₉₆₇₎ Semen _{output of rabbitsejaculated} after_{varyingsexual}

preparation.Proc.Soc._exp.Biol. Med.125, 1278.

Resko, J.A. &_Eik-Nes,K. B.₍₁₉₆₆₎ Diurnal testosteronelevelsin_{peripheral plasma}of human male

subjects. J.clin. Endocr.26,573.

Saginor,M. &Horton, R. ₍₁₉₆₈₎ Reflex release of_gonadotropinand increased_plasmatestosterone concentration inmalerabbits_{during copulation. Endocrinology, 82,627.}

Taleisnik, S., Caligaris, L. &Astrada,J. J. (1966) Effect ofcopulationontherelease ofpituitary