Dissociation of increases in plasma insulin-like growth factor I and testosterone during the onset of puberty in bulls

Dissociation

of

increases

in

_plasma

insulin-like

_growth

factor

I

and

testosterone

_during

the

onset

of

_puberty

in

bulls

R.

_Renaville,

S.

Massart,

M.

Sneyers,

M.

Falaki,

N.

Gengler,

A.

_Burny

and

D.

Portetelle

Department

of

Molecular

_Biology

andAnimal

_Physiology;

and

_Department

_of

Microbiology,

Faculty

of

Agronomy,

2,

_Passage

des

_Déportés

B-5030 Gembloux,

Belgium

The

_present

_study

was conducted to examine the

relationship

between

plasma

concen-trations of testosterone, insulin-like

_growth

factor I

_(IGF-I)

and

_IGF-binding

_proteins

(IGFBPs)

_{during puberty,}

in male calves treated with GnRH or testosterone

_propionate.

Twelve male Holsteincalves

(10

weeks

old)

were

assigned

to the control _group

_(n

=

6),

the GnRH-treated _group

(n

=

3)

or the testosterone-treated _group

_(n

=

3).

For 8

weeks,

the

GnRH-treated_group receiveda

single

i.v.

injection

ofGnRH

(0.5

\g=m\g

kg

\m=-\1

body

mass)

each

day

while the testosterone-treated _group received an i.m.

_injection

of testosterone

propionate

(0.5

mg

kg

\m=-\1

body

mass)

twice a

day.

The calves werestudied until

they

were

200

_days

old. Hormone treatments were

stopped

one month after

puberty

was reached in

the control _group. Blood

_samples

were collected _every 30min for 8 h every third

day.

Hormone concentrations were determined

by radioimmunoassay.

Western

ligand blotting

and

_{immunoblotting,}

_using

monoclonal antibodies

_against

IGFBP-2 and

IGFBP-3,

wereused

to characterize the

_IGF-binding

_proteins.

In the control _group,

_puberty

occurred at about

120

days

ofageandwas associated withanincrease inconcentrations of testosterone, IGF-I and IGFBP-3 and a decrease in concentration of IGFBP-2. In the GnRH-treated _group,

plasma

testosterone remained low until 8 weeks after establishment of

_puberty

in the control _group

(4

weeks after the end

of treatment).

In the testosterone-treated _group,

testosteronewas

high

during

thetreatment

period

and then decreased to

_prepubertal

values when treatment was

stopped.

Testosterone values increased

_again

to reach

postpubertal

values 5 weeks after the end of hormone treatment.

Nevertheless,

_independent

of

testosterone status, the

_profile

of IGF-I and the IGFBPs in the GnRH- and testosterone\x=req-\

treated _groups were

parallel

to that

reported

for the control _group with the transitionfrom

prepubertal

to adult values at about 120

_days

of _age. In

_conclusion,

concentrations of testosterone, IGF-I and IGFBP-3 increase

_together,

but

_{probably independently,}

_during

the

onset of

_puberty

in male calves.

Introduction

The onset of

_puberty

has been shown to be a

complex

synergism

betweensexsteroids and insulin-like

growth

factorI (IGF-I) in humans

_(Metzger

et al, 1994),

_sheep

_(Lord

et

al,

1991) andin cattle

(Renaville

et al, 1993).

_During

this

_period,

testosterone isreleased

_episodically

and

_plasma

concentrations

of IGF-I increase

_strikingly,

concomitant with an increase in

mean

plasma

concentration of testosterone. A

_positive

corre¬

lation was noted between the concentrations of these two hormones for bull calves

_during

this

_{physiological}

_step

(Renaville

et

al,

1993). Moreover, as in humans

(Smith

et

al,

1993), baboons

(Crawford

et al, 1994) and

_pigs

(Lee et

al,

1991a), in

_cattle,

the transition from

_prepubertal

to mature

male is characterized

_by

a marked increase in IGFBP-3

concentrations

(Renaville

et

al,

1993).

However, information on the

possible

link between IGF-I

and testosterone

_synthesis

_during

this

_{physiological}

_step

is

contradictory. Jasper

(1985), Harrisetal (1985) andAttie etal

(1990),inhumans, andLeeelal

_(1991b),

incattle,

_reported

that sex steroids stimulate IGF-Iconcentrations

directly

at thetime

of

_puberty,

while Crawford et al (1993) concluded that the

pubertal

IGF-I _surge in mice does not

_require

_androgens

in

either the _pre- or

postpubertal periods.

In addition,

Godfrey

et al (1992) demonstrated that treatment with testosterone

propionate

delays

puberty

and

_{endogenous pulsatile}

release of testosterone in bull

calves,

while

_Ronayme

et al (1993)

described an increase in testosterone concentrations in

prepubertal

bulls treated

_continuously

with GnRH.

In the

_study

described here,

_prepubertal

bull calves were

treated with testosterone

_propionate

or GnRH to determine whetherIGF-Iis

dependent

onthe

androgen

_surgeat theonset

of

_puberty.

Materials and Methods

Animals

Twelve,

_spring-born

(identical

calving day),

Holstein male

calves₍₁₀weeks

_old)

were

assigned

toeitherthe control _group

(n=6) _or

groups treated with either GnRH (n=3) or testos¬ terone

_propionate

(n=3). The 6x3x3

experimental design

used in the

_present

_study

is

justified

_by

the fact that all data have been evaluated

by

comparison

with the_age of theonset

of

_pulsatile

testosterone releaseinthe control group.The

large

number of animals in _{the control group} was

judged

_necessary

tocharacterize this

_{physiological}

_stage

_exactly.

Animals were fed with _grass

hay

twice a

day

and a

commercial allmash

_containing

130_gcrude

_protein

_kg

~

1.

Feed

intake was controlled so that

body

mass

gain

was about

950_g

day

~ ; water was

supplied

adlibitum. All animalswere

kept

inidentical conditions.

_Daily

room

temperature (18°C)

and

daylength

(16h

_light:8

h

_dark)

wereconstant.Initial meanlive masses were 74.4±4.8

kg

for the controlgroup, 75.9±3.2

kg

for the GnRH-treated _group and 73.1±5.9

kg

for the

testosterone-treated _group.

Experimental

design

Fora

period

of8weeks, the GnRH-treated group receiveda

daily

i.v. injection of GnRH _{(0.5 µg}

_kg-

_body

mass;

Sigma,

St Louis, MO) in 0.5ml saline and the testosterone-treated

group was

given

an i.m. injection of testosterone

propionate

(0.5mg

kg

~

.

body

mass;

Sigma)

in 0.5 ml saline twicea

day.

Control calves receivedani.m.injectionof saline(0.5

ml)

daily.

Animals were studied between 70 and 200

_days

of _age

(day

0=

birth).

Hormone treatments _were

stopped

_onemonth after

puberty

wasachievedinthe control_group.

Puberty

wastaken as the time when more than two successive values of testos¬ terone were greater than 2_ng ml~ (Renavilleet al, 1993).

Blood

_samples

₍₅

_ml)

were taken from the

jugular

vein viaa

polyurethane

catheter into

_heparinized

tubes at 30 min inter¬

vals from 08:00h to 17:00h _every third

_day.

_Samples

were

stored

_immediately

at 4°C and then

_centrifuged

_{at 1800 g} for

10 min.Plasmawas

separated

and storedat

—

20°Cuntilitwas

used in the hormone and

_{binding-protein}

_assays.

Iodination

_procedure

The recombinant bovine IGF-I

_(rbIGF-1;

batch GTS-1) used as iodinated tracer (in

_{radioimmunoassay}

and western

_ligand

blotting)

and as standard (in

radioimmunoassay)

was

kindly

donated

_by

Monsanto (St Louis, MO). The hormone was

labelled with _{Na[ I]}

_by

the chloramine method

_(Parker,

1990).

I25I-labelled

rbIGF-I was

separated

from free iodine

using

a Centricon-3microconcentratorasrecommended

by

the

manufacturer (Amicon Division,

Beverly,

MA). The

_specific

activity

was 70

_pCi

_pg~

.

Tritiated testosterone ([1,2,6,7- Hjtestosterone, batch TRK 402,

_specific

activity 80-105 Cimmol~

J)

was

purchased

from

Amersham International

_(Amersham,

Bucks).

Production

_of

monoclonal antibodies

_against

bovineIGF

_binding

protein

(IGFBP)

-2 and-3

Anti-bovine IGFBP-2 and IGFBP-3 monoclonal antibodies

were

produced

inour

laboratory

(Porteteile

etal, 1995).

Briefly,

ten-week-old female BALB/c mice were immunized with

100 _µg recombinant fusion

_protein

bovine IGFBP-2 (or

-3)-maltose

_binding

_protein

(MBP) in

_incomplete

Freund's

adju¬

vant (0.10ml total

_volume)

_equally

_distributed into the rear

footpads.

Fourteen

_days

after immunization,micewerebledas

recommended

_by

Mirza et al (1987) and the

_draining

_lymph

nodes

_(popliteal

and

_inguinal)

were removed and

pooled

for fusionas described

by

Brücketal. (1982)with themodification that NSO murine cells wereused as

myeloma

partner.

Specific antibody-producing

hybridomas

were identified

by

differential indirect

_{enzyme-linked}

immunosorbent assays

(ELISA) tests

_using

recombinant

_proteins

IGFBP-MBPorMBP adsorbed onto the wells

(Porteteile

et

_al,

1989). Positive

hybridomas

wereclonedtwiceinsoft_{agar, grown}on

Pristane-primed

BALB/c mice and then

_purified

by

ion-exchange

chromatography.

In the

_present

_study,

the 7B5 anti-IGFBP-3

(isotype IgGl)

monoclonal

_antibody

and the 8G10

anti-IGFBP-2

_{(isotype IgGl)}

monoclonal

antibody

wereused inthe

immunoblot studies.

Hormonedetermination

Testosterone was determined

by radioimmunoassay

as

described

_by

Renaville et al (1983). The minimum detectable dose of testosterone was 0.1_ng ml-1. The intra- and inter¬ assay coefficients of variation were 4.8% and 10.7%, _respec¬

tively,

for the low standard concentration (0.8ng ml~ ) and

2.1% and 6.1%,

_{respectively,}

for

_high

standard concentration

(10ng

ml-1).

The

_{radioimmunoassay}

for IGF-I in bull

_plasma

was _per¬ formed

_according

to the method described

_by

Lemal et al (1989)and

_adapted

_by

Renavilleetal_(1993).Inthismethod,as

reported

by

Breier etal (1991),a

cryoprecipitation step

isused

to eliminate

_aggregated

_proteins

in

_plasma

extracts.

_Briefly,

after acid-ethanol extraction (87.5% ethanol and 12.5% HC1

mol 1~

v/v),

an

aliquot

of the

supernatant

was neutralized

with 0.855mol Tris base 1~'

at a ratio of 5:2. The

_samples

were storedat

—

20°C for 1hand then

_centrifuged

_{at 3000 g}

for30 min at4°C.The

_supernatant

wasdecantedintofreshtest

tubes and used in the

radioimmunoassay.

The minimum

detectable dose of IGF-I was 1_ngml~

. Intra- and interassay coefficients of variation were 12% and 16%,

respectively,

for the low standard concentration (25 ng

ml-1)

and 6.5% and

9%,

_{respectively,}

for the

_high

standard concentration

(250ng ml"

x).

Western

_{ligand blotting}

Western

_{ligand blotting}

was

performed

to evaluate

plasma

IGFBPs

_following

the method described

_by

Renaville et al

(1993).

_Briefly,

1

_µ

of SDS-denatured

_plasma

was

applied

to a

4%

_{stacking gel}

and

_{electrophoresis}

was

performed

through

a

12.5%

_{polyacrylamide gel.}

Prestained

I4C-labelled

molecular

weight

markers

(Amersham)

were runin

parallel

lanes.The

gels

were then soaked in Towbin buffer (25 mmol Tris 1~

\

192mmol

_glycine

1

_\

20%

_(v/v)

methanol,

_pH

8.3) and

proteins

were blotted onto nitrocellulose sheets

(Hybond-C,

Amersham).

Electrophoresis

and electrotransfer were _per¬ formed

_using

theMini-Protean II system

(Bio-Rad, Richmond,

CA). After saturation, membranes were incubated with

I25I-labelled

_{rbIGF-I (4000 c.p.m.} cm "

2

blot)

overnight

at4°C.

The membranes were then

washed,

air-dried and

exposed

_to

Kodak X-Omat AR films

(Rochester,

NY) for 2-4

_days

at

-70°C.

Autoradiograms

werescanned

_using

a

Sharp

JX325 scanner and band intensities were

analysed

with imagemaster id soft¬ ware

(Pharmacia,

Uppsala).

A

plasma pool

wasusedas internal

standard.

Immunoblotting

Plasma

_samples

_(1.5

_{µ )}

were denatured

by

SDS with

di-thiothreitol as

reducing

agent

and then

applied

to a 4%

stacking gel;

electrophoresis

was

performed through

a 12.5%

SDS-polyacrylamide

gel,

and blotted onto Immobilon sheet.

Electrophoresis

and _{electrotransfer} were

performed

using

the

Mini-ProteanII

_System

_(Bio-Rad).

Membranesweresaturated for1hatroom

_temperature

with a 10% defatted milk solution in Tris saline buffer (10mmol Tris-HCl 1

"

\

150mmol NaCl

\

pH

7.4),

and _then incu¬

bated

_overnight

at 4°C with monoclonal

antibody

(mAb)

7B5

(2.4pg

mP1)

or mAb 8G10 (2.1_{pg ml"}

:)

diluted in Tris

saline buffer

_containing

0.1%

_(v/v)

Tween20.Membraneswere

washed threetimesinTris saline buffer

_containing

0.1%Tween

20

_(v/v),

followed

by

incubation for 1h at room

_temperature

with alkaline

_phosphatase

_coupled

_{goat-anti-mouse}

_antibody

(0.133pgml" ;

Promega,

Leiden)

inTris saline buffercontain¬

ing

0.1%Tween 20

(v/v).

Membraneswerewashed four times

in Tris saline buffer

_containing

0.1% Tween 20, and

alkaline-phosphatase

activity was revealed

_using

NBT/BCIP substrate

(KPL,

_{Gaithersburg,}

MD).

Immunoblot membraneswere scanned

_using

a

Sharp

JX325

scanner and band intensities were

analysed

with imagemaster

id software. A

plasma

pool

was used as the internal

standard.

Statistical

_analyses

Valuesare

presented

as means±sd.

Between-group

signifi¬

cancefor

parameters

wasassessed

by analysis

ofvariance

_using

the GLM

_procedure

of sas software

(SAS®,

1991). The

following

model wasused:

#

=

µ+ ,

₊

,

+

_{( ),>}

+_^

where

_Y¡jk

=observations for

dependent

variables of_treatment

i

_{during period}

jonanimal

k;

_µ=overall_mean;

T¡

=_treatment

effect

_(i

=_{1, 2,} 3);

P¡

=

period

effect(

j

=1, 2, 3.41, 42, 43);

( )1(

=fixed effect associated with interaction between treat¬

ment i at

_period

_;',·

andeik=random residual effect for the ith

treatment and the

_jth

_period.

Significant

differencesamongmeans weredetermined

by

the

Ftest at 5%

_probability.

Results

Changes

in theconcentrations

_of

hormones

_during

_puberty

In thecontrol group, mean

plasma

concentrations oftestos¬ terone increased

_{significantly}

(P<_0.001) from 105 ±5

_days

old _(mean value: 0.7+_{0.2 ng}

_ml"1)

_{to 120±10}

_days

(2.1±0.3ngml~

J)

(Fig.

la).

During

this

period,

release ofthe

hormone became

_pulsatile

_{(values ranging}

from 0.2±0.1 ng

ml~

1

to 4.3± 1.9_ng ml~

:), indicating

that

_puberty

had been reached. Mean concentrations of testosterone then increased

progressively

to

day

160(meanvalue:3.1±0.8ngml~

:),

after

which

_they

remained

_relatively

constant_{up to} the end of the

experimental period.

Hormone treatments had different effects on

plasma

testos¬ terone concentrations. Injections of testosterone

_propionate

immediately

induceda

significant

(P<_0.001)increase in

_plasma

testosterone;

_however,

therewas no

daily

episodic

release,and

concentrationsremained

_higher

(7—9ngml~ ; <0.001) than

in the control group

during

the treatment

_period

_(Fig.

la).

When testosterone treatmentwas discontinued

(day

150;one

month after the onset of

_puberty

in the control

_group),

mean

plasma

concentrations fell to

_prepubertal

values _(0.5±0.2_ng

ml

~ )in

one

week,

and werelower than inthe control group

(P< 0.001)

_(Fig.

la). Itwas

only

at 187±5

days

(33± 7

days

after the end of treatment) that a

pulsatile

release of

endog¬

enous testosteronewasosbervedin this_{group and} mean

daily

concentrations reached similarvalues to those inthe controls.

In the GnRH-treated_group,a

daily

injectionofGnRH affected the release of testosterone. From

_day

70 to

_day

110, mean

values were low

(range

values: 0.3—0.7_ng

ml-1)

and no

significant

differences wereobserved with the control _group. After

day

110, whiletestosterone concentrationsinthecontrol groupincreased,valuesintheGnRH_group,remained low until

day

163 ± 6(3.1 ±0.8_ngml~~ inthe control_group

_compared

with 0.2±0.1 _ng ml"1 in the _{GnRH group;} <0.001), from

which time concentrations

_slowly

increased,

_finally

_exhibiting

pulsatile

release and similar mean values to the control _group

(P>0.05)at

_day

181±4.

During

the

_{experimental period,}

mean

plasma

concentrations

ofIGF-I exhibited asimilar patterninall _groups. From

_day

70 to

_day

_110, mean

plasma

IGFT values were

relatively

constant and no

significant

differences (P>0.05) were observed

between the

_experimental

_{groups and the control group,} with mean values for the

period

as follows: control _group: 93+ 14_ng

_ml"1;

testosterone-treated _group:

_76±17ng

ml":

and GnRH-treated group: 71±19_ng ml"1

(Fig.

lb).

From

_day

110 to

_day

130, a

sharp

increase (P<0.001) in

plasma

concentrations was observed in all _groups:

182±35ng ml~

:

in the control _group; 156±24_ng ml~

:

in

the testosterone-treated group and 163+_{34ng ml}

~ in the

GnRH-treated _group. IGF-I concentrations in the control

group then continued to increase, to reach values of about

250-300_ng ml~

l

at the end of the

_experiment.

In the two

treatment groups, IGF-I concentrations increased more

slowly

and were below values for the control _group between

days

145 and 155 (P<_0.01). A second increase was observed between

days

160 and 185 in the two treatment groups

when values similar to those seen in the controls were reached.

Fig. 1.Mean

_plasma

concentrations of(a) testosteroneand(b) insulin-like

_growth

factorIin(O) control, (·)

testosterone

_{propionate-treated}

and ( ) GnRH-treated bulls

_during

the onset of

_puberty.

(D) The

_period

of

treatment.Arrowsindicate theonsetof

_puberty

ineach_group: (O) control; (·)testosterone

_{propionate-treated}

and ( ) GnRH-treated _groups.

Western

_{ligand blotting}

_of

IGFBPs

Western

_{ligand blotting}

of bovine

_plasma

revealed four distinct bands that

_specifically

bound

125I-labelled

IGF-I.Their molecularmasseswere,

respectively,

45-54, 38,28and24kDa.

Inthecontrol_group,theonsetof

_puberty

wascharacterized

by

an increase from

_day

100 (0.69+_{0.14 units) to}

_day

₁₅₀

(1.72±0.21 _{units) (P}<0.001) in the

_intensity

of the doublet

45—54kDa band, which was identified as IGFBP-3, after which it remained constant until the end of the

_experiment

(Fig.

2a). The

_intensity

of the IGFBP-2 ₍₃₈

_kDa)

band was

markedly

reduced from

_day

120(0.67±0.18 units)to

_day

160

(0.17±0.08 _{units) (P}<_0.001) and remained low until the

end of the

_experiment

_(Fig.

2b).

During

this

_period,

no

Fig.2.Mean

_plasma

concentrationof(a)insulin-like

_growth

_{factor-binding}

_{protein-3 (IGFBP-3)}and(b)IGFBP-2

in(O)control, (·)testosterone

_{propionate-treated}

and( ) GnRH-treated_groups

_during

theonsetof

_puberty.

( ):The

_period

oftreatment.Arrowsindicate theonsetof

_puberty

in_{each group:}(O)control;(·)testosterone

propionate-treated

and( ) _{GnRH-treated groups.}

corresponding

toIGFBP-1 and_IGFBP-4,

_respectively

_(data

_not

shown).

Daily

injections oftestosterone

_propionate

orGnRH hadno effecton mean

plasma

concentrations ofIGFBP-2andIGFBP-3.

Indeed,

the

_profile

of these two

_proteins

was similar to that

reported

for the control_group, with a

progressive

increase in

IGFBP-3 fromabout

_day

100 and a

sharp

decreasein IGFBP-2 after

_day

120 in both _groups. No

_significant

difference

(P> 0.05)wasobserved on_any

day

of collection between the values in controland treated groups.

Immunoblotting

of

IGFBP-2 and IGFBPS

The immunoblot

_{method, using}

monoclonal antibodies

against

bovine IGFBP-2 and IGFBP-3,showedasimilar

_pattern

Fig. 3. (a) Plasma insulin-like

_growth

factor

_binding

_protein-3 (IGFBP-3) and(b) IGFBP-2 inanindividual bull calf3 weeks before(lanes1-3) and three weeks after(lanes5-7)

puberty.

Lane4 _representsthe week

during

which the animal attained

_puberty

(pulsatile

release of testosterone).The _proteins were revealed

usingmonoclonalantibodies against bovine IGFBP-3 or _IGFBP-2.

for these two

_proteins

_throughout

the

experimental period

(Fig.

3)andwasin

_agreement

with dataobtained

_using

western

ligand blotting.

Discussion

The

study

reported

herewasconducted to define the relation¬

ship

between

_plasma

concentrations oftestosterone and IGF-I

during

puberty,

by

treating

male calves with GnRH or testo¬

sterone

_propionate.

In the control group, theonset of

_puberty

in bull calves was identified

by

concomitant rises of

plasma

concentrations of testosterone and IGF-I, as described

by

Ronge

and Blum (1989a,

b)

and Renaville et al (1993). It has

been

_{suggested by}

_previous

studies,

particularly

in humans

(Kerrigan

and

_Rogol,

1992; Zachmann, 1992;

_Metzger

et

al,

1994),that these coordinatedhormonalincreasesaredriven

by

growth

hormone. Indeed, data from the control _group in the

present

study

suggests

that the

_{puberty-associated}

increase in

plasma

concentrations of IGF-I could result from the stimula¬

toryeffect oftestosteroneonthe

somatotrophic

axis,

indirectly

mediatedvia the

_oestrogen

_receptor

after thearomatizationof

testosterone to oestradiol. Schwarz et al. (1992)

_reported

that

gonadectomy

incattlecauses afall in

plasma

concentrationsof

GH, testosterone and IGF-I and Metzer and

_Kerrigan

(1994) showed that

_blocking

the

_oestrogen

_receptor

with tamoxifen results indecreased 24h meanserum GH and

plasma

IGF-I in

humans. _However,

_Metzger

et al. (1994) concluded that it is

difficult to differentiate between the testosterone effects because the hormone can act either

_directly

_through

the

androgen

receptor

or

indirectly

through

the

oestrogen

receptor

once it is aromatizedto

_oestrogen.

Under the

_experimental

conditions in the

_present

_study,

hormonal treatment of

_prepubertal

bulls inhibited

_endogenous

testosterone

_production.

The

_partial

_suppression

of

_gonadal

steroidogenesis

by

GnRH _(a

_prepubertal

levelwas

maintained)

observedin this

study

are in

_agreement

with

_{previous reports}

in men (Harris et al, 1985;

Styne

et al, 1985), rams

(Lincoln

et

al,

1986) and

_{stags (Lincoln,}

1987) but are in conflict with

studiesin maturebulls

_(Rechenberg

etal, 1986;

_Ronayme

et

al,

1993). Mannet al. (1984)

_reported

that chronicadministration of

_agonist

_analogues

ofGnRH inducedadesensitization ofthe

pituitary

gonadotroph

in rhesus

monkeys.

These authors also

reported

that infusion of a GnRH

agonist

in newborn rhesus

monkeys

suppressed

the

_{expected prepubertal}

increase in LH

and testosterone (Mann et al, 1989). In rats, the

_postnatal

increase in testosteronecould be abolished

by

treatment with

either GnRH

_agonist

or

_{antagonist (Kolho}

and Huhtaniemi,

1989). Several studies have also

_emphasized

the

_relationship

between testosteroneand avariety ofsteroid

growth

promot¬

ers.Itwasobserved

(Fabry

et

al,

1983, 1984;Joneset

al,

1991;

Godfrey

et al, 1992) that the

_pulsatile

nature of testosterone

profiles

wasabolished, testosterone secretion

markedly

reduced

(but

not

suppressed)

and

puberty

delayed

aftertreatment with

androgen,

progestagen

or

_oestrogen

aloneor in combination.

In the

_present

_study,

the

_sharp

testosteronedecrease observed in the testosterone-treated _group after the end of treatment

confirmed that

_endogenous

testosterone

_synthesis

was at a

prepubertal

level.

There are few studies that have examined the effects of

testosterone or GnRH administration on the

production

ofIGF-Iin

prepubertal

males. Schwarzetal.(1992)observedno difference in

plasma

IGF-I

profiles

between bulls castrated

before

_{puberty (gonadectomy}

at 108

_days

of

_age)

and intact

bulls until

_{approximately}

250

days

ofage. In the

present

trial,

although

major inhibition of the secretion of

_endogenous

testosteronetook

_place

_during

both hormonaltreatments,there was no major alteration in IGF-I

profiles during

the onset of

puberty

in thetwo treatmentgroupswhen

compared

with the

controlgroup. The aim of the

_present

study

was to determine

by

which

_pathways

IGF-Iwasmodified

by

GnRH ortestoster¬

one treatment. The first observation in the

_present

trial was

that, contrary to the situation in castrated

animals,

the inhi¬ bition of

_endogenous

testosterone

_synthesis

in theGnRH- and testosterone-treated _groups was not

_complete.

Testosterone

the IGF-I surge at

puberty.

However, since testosterone

treatmentdidnotinducean increaseinIGF-I,it is

_possible

that

the_{enzymesnecessary} to aromatizetestosterone to

_oestradiol,

which is known to induce a

growth

hormone-induced IGF-I

response in cattle (Breier et

al,

1988), are

only

present

at

puberty.

Another

_possibility

was

suggested

by

Hobbs _et al (1993) who observed different _effects of treatment with nan-drolone and testosterone enanthate on IGF-I

synthesis

by

a

qualitative

difference in

_androgenic

_potency

attheneuroendo¬

crine

_level,

_particularly

if local 5 -reduction of the

_androgen

occurs. However, Tenover and Matsumoto (1992) found no

change

in either

_growth

hormone or IGF-I values in young

adult men treated with testosterone enanthate for 12 weeks.

Low IGF-I values from

_day

145 to

_day

155 in the treated

groups could also indicate that testosterone

_synthesis

was

insufficient to sustain normal IGF

_production

via

_oestrogen

receptors

and stimulation

_by

_growth

hormone, as

reported

in steers

by

Schwarz et al. (1992). This

_hypothesis

is also

supported

by

the IGF-I increase noted in the

_present

study

after the

_{physiological}

induction of a

pulsatile

testosterone

release in both treatment_groups.

IGFs in

_plasma

and most

_body

fluids are

complexed

with

high-affinity binding

proteins

(IGFBPs). Numerous studies on nutritional status or treatment with

growth

hormone have

clearly

demonstrated thatIGFBP-3, the

_predominant

IGFBP in

plasma,

isrelevantto the

_{pharmacokinetics}

andactionofIGF-I,

while there is a

negative

correlation between IGFBP-2 and

IGF-I concentrations

(Cohick

et

al,

1992; Thissenet

al,

1994).

Taking

into account this

information,

the results of our

previous

study

(Renaville

et

al,

1993)andthe data observed

_by

Lee et al

(1991b)

in

_pigs,

the transition from

_puberty

to

maturityin bullcalves _maybe identified

by

amarkeddecrease

in IGFBP-2 and

_by

an increase in IGFBP-3.

This is the first

_report

of the use of

specific

monoclonal

antibodies

_against

bovine IGFBP-2 and IGFBP-3 in immuno¬

blotting

to confirm the

_changes

in

_plasma

concentrations of

IGFBP-2andIGFBP-3 observed

_by

western

_{ligand blotting.}

As

wasobserved forIGF-I,

plasma

concentrationsofIGFBP-2and

IGFBP-3intreatedanimalsfollowed the

_profile

_reported

forthe control _group and

_appeared

unaffected

_by

testosterone status. In conclusion, our

study

shows that the abolition of the

pubertal

rise in

_plasma

testosterone,

by

treatment with GnRH

ortestosterone

_propionate,

does not have a marked effecton

IGF-Ioronits

plasma-binding

proteins

(IGFBP-2andIGFBP-3) in male calves. However, our resultssuggest that testosterone

can still stimulate IGF-I

synthesis,

as observed in both treat¬ ment _{groups between}

_days

180 and 190, when

_endogenous

plasma

concentrationsoftestosteroneare increased.Wethere¬ fore consider: (1) that the

_pubertal

_androgen

_surge is not the

majorfactor

_responsible

forthesimultaneous increase in IGF-I and (2), that testosterone and IGF-I values increase simultane¬

ously,

but

_{individually,}

attheonsetof

_puberty

inbulls. Further

investigations

are needed to understand the mechanism

_by

which the

_synthesis

of IGF-I and testosterone are induced at

puberty.

Financialsupport for this

_study

was

provided

by_{theInstitut pour}

l'Encouragement

de la Recherche

_Scientifique

dans l'Industrie et

l'Agriculture

(research grants 5512 A and 5645 A) and

_by

Pôle d'Attraction Interuniversitaire research _grant PAI 15. The authors

wish to thank Y. Braet, R-M. Maistriaux and G. Tavernierfor their technical assistanceandJ.Moremanfor her

_help

inthe_preparationof the _manuscript.

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