Physiological studies on the sex-linked dwarfism of the fowl : a review on the search for the gene's primary effect.

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Physiological studies on the sex-linked dwarfism of the

fowl : a review on the search for the gene’s primary

effect.

M. Tixier-Boichard, L.M. Huybrechts, E. Kühn, Eddy Decuypere, J. Charrier,

P. Mongin

To cite this version:

Review article

Physiological

studies

on

the sex-linked dwarfism

of the fowl:

a

review

on

the search for the

_gene’s

primary

effect

M.

Tixier-Boichard

L.M.

_Huybrechts

E. Kühn

2

E.

_Decuypere

₃

J.

Charrier

P.

_Mongin

1 _{Institut National de la Recherche}

Agronomique,

Laboratoire de

_{Génétique Factorielle,}

78350

Jouy-en-Josas,

France;

2

_{Zoological Institute,}

Laboratory

of

_{Comparative Endocrinology,}

Naamsestraat 61, 3000 Leuven,

Belgium;

3

_Laboratory

for

_Physiology

of Domestic Animais, Kardinaal Mercierlaan 92, 3030 Heverlee,

Belgium;

4_{Institut National de la Recherche}

_Agronomique,

_{Station de}

_Physiologie

_{de la Croissance, 9}

_place

Vala, 34060

_{Montpellier, France;}

5 _{Institut National de la Recherche}

_Agronomique,

_{Station de Recherches Avicoles, Centre de} Recherches de

Nouzilly,

37380 Monnaie, France

(received

31

_May

1988,

accepted

26

_September

1988)

Summary —

The results obtained in the last 10 yars on the

_{endocrinological}

and metabolic effects

of the sex-linked dwarf _gene of the fowl are reviewed in order to

_identify

its mode of action on

_growth

and other traits.

Among

the main factors involved in

_{growth regulation, thyroid}

hormones, T4 and T3,

growth

hor-mone, GH, and its related

growth

factor, IGF-I, were the most studied in dwarfs. These birds are characterized

by

low

_circulating

levels of T3 and IGF-I in

_spite

of normal or even increased levels of T4 and GH. The T3

_deficiency

is

explained by

a lower

peripheral activity

of T4 monodeiodination which could be related to an abnormal T4

_{uptake by}

the _{cell, particularly} the

_hepatocyte.

The low

production

of IGF-I could be related to a deficient GH receptor, as

_{suggested by}

the decreased GH

binding

observed in the liver of dwarf birds. Both T3 and IGF-I

_synthesis

_{may share} common

path-ways since

thyroidectomy

also decreases IGF-I level while a GH

injection

stimulates the T4 to T3 monodeiodination in the normal

_embryo

but not in the dwarf. Further studies are needed on the GH

receptor and the T4

_uptake

in the

_hepatic

cell to

identify

the common

_point

where the _{dwarf gene} could act. Ovulation rate and

_{lipomobilisation}

are decreased in adult dwarfs but these

_findings

are

not _yet

_easily

related to the

endocrinological changes

observed

_{during growth.}

A

_{complementary approach}

to _compare the _genotypes would be to study the DNA

_polymorphism

using

available molecular

probes

sex-Ilnked gene - dwarfism - chicken -

endocrinology

Résumé —

Synthèse

des études

_{physiologiques}

du nanisme Ilé au sexe chez la

poule

en _vue de la recherche de l’effet

_primaire

du

_gène.

La

_{récapitulation}

des résultats obtenus

_depuis

dix

ans sur les effets hormonaux et

_{métaboliques}

du

gène

de nanisme lié au sexe chez la

poule

Parmi les

_principaux

facteurs endocriniens affectant la croissance, les hormones

thyroidiennes,

T4 et T3, l’hormone de croissance, GH, et le facteur de croissance IGF I ont été les

_plus

étudiés chez les nains. Ces animaux montrent de faibles taux circulants de T3 et d’IGF

_malgré

des taux nor-maux voire augmentés de T4 et de GH. La diminution de T3

_s’explique

_par une moindre monodeio-dination

_{périphérique}

de _T4,

_probablement

reliée à une

_{disponibilité}

insuffisante de T4 dans la

cel-lule

hépatique.

La diminution d’IGF

pourrait

être

rapprochée

du déficit en

_récepteurs

de GH,

expérimentalement

démontré chez les nains. Les

_synthèses

de T3 et d’IGF

_{I pourraient}

_partager

des voies communes car la

thyroidectomie

diminue aussi l’IGF circulante alors _qu’une

_injection

de

GH stimule la monodeiodination de T4 en T3 chez

_l’embryon

_{normal, mais pas} chez le nain. Les

études du

_récepteur

à GH et des mécanismes d’entrée de T4 dans

_{l’hépatocyte}

_{doivent être}

pour-suivies pour

préciser

le

_point

commun où

_pourrait

intervenir le

_gène

de nanisme. La diminution du taux d’ovulation et de la

lipomobilisation

observée chez l’adulte nain est encore mal

_expliquée.

Une

_{approche complémentaire}

_{pour comparer} les

_génotypes

serait d’étudier le

_{polymorphisme}

de

I ADN avec les sondes moléculaires

disponibles.

nanisme -

_gène

lié au sexe -

poulet - endocrinologie

Introduction

The sex-linked mutant _gene

_{(d! causing}

dwarfism in the fowl

_(Gallus

domesticus)

was

first described

_by

Hutt

_(1959).

It has been shown to occur in different

_populations

around the

world,

as reviewed

_by

Guillaume

_(1976).

It has been

_generally

assumed that dwarf mutations of different

_origin

_correspond

to the same _gene. A

_large

number of

_genetic,

zootechnical and

_{physiological}

studies have

_already

been carried out that have led to the commercial use of the mutation in the maternal lines of the broiler

_industry.

However,

the mode of action of the gene has not

_yet

been

_precisely

defined. Such

_knowledge

would

have both

applied

and fundamental consequences. Practical consequences would be to

determine the effects of the gene on other traits such as ovulation rate.

The identification of the

_{protein directly}

modified

_by

the gene would

provide

a basis for the molecular

_cloning

_{of the gene:} a

_probe

for the sex-linked dwarf mutation would be

useful to compare the different mutants known in the world and

eventually

to _{prove their}

identity. Cloning

the gene would also

provide

a tool to

_genetically

_manipulate

_growth

in birds. From a more

_{general point}

of

view,

dwarfism can also be used as a model to

understand

_{growth regulation.}

This review will

_mainly

deal with the results of the last 10 years that have been ob-tained on

_{endocrinological}

and metabolic _{effects of the gene. The main factors involved} in

_growth

_regulation

will first be

_presented

in the normal

chicken,

then the modifications observed in the

_growing

dwarf chick will be described. A

_synthesis

of these modifications may

help

to derive a

_working

_hypothesis

on the

_{probable primary}

effect of the _gene. Some data obtained in the adult dwarf hen will then be

_briefly

considered in connection with the

physiological changes

which are known in the dwarf chick.

Finally,

further

experi-ments aimed at

_testing

our

_hypothesis

can be

_considered,

in the field of

_physiology

and

perhaps

also of molecular

_biology.

Hormones involved in

growth

in chicken

According

to Scanes et al.

_(1984),

_growth

hormone

(GH)

on the one hand and

thyroid

for the normal

_growth

of a chick. Growth factors are

_being

increasingly

_studied,

such as

insulin-like

_growth

factors I and

II,

IGF-I

being

also called

_{somatomedin-C,}

_epidermal

growth

factor

_(EGF),

nerve

_growth

factor

_(NGF)

or vitamin D metabolites. Other

_important

hormones such as

insulin,

glucocorticoids

or steroids also

_{play a}

role in

_growth

regula-tion. The

_synthesis

and release of GH

_by

the

_pituitary

is

_{regulated by hypothalamic}

pep-tides,

in a

_stimulating

manner for

_growth

hormone

_releasing

factor

(GRF)

and

thyrotro-phin

releasing

hormone

_(TRH),

in an

_inhibitory

manner for somatostatin

_(SRIF).

The roles of vaso-active intestinal

_peptide

_(VIP)

and

_prolactin

remain to be clarified so far as

growth

is concerned. These different hormones and

_growth

factors have been

_presented

in

_Figure

_1,

with their

_{relationships}

and their sites of

_synthesis.

Some comments _appear

particularly

relevant in view of a

_study

on _{the dwarf gene: the}

_stimulating

role of human GRF and TRH on GH secretion has been shown in several mammalian

_species

and in chicken. Growth hormone elicits the

_synthesis

of IGF-I in the rat liver

_(Schalch

et

_al.,

1979;

Mathews

_{et al.,}

_1986),

but other tissues are also able to

_produce

IGF-I. In the chic-ken it has

_recently

been demonstrated that an intravenous

_injection

of GH was also able to increase the

_plasma

concentration of IGF-I

_(Huybrechts

et al.,

1988).

The chicken

thy-roid

_gland

_mainly

releases

_{tetraiodothyronine (T4)}

in the

bloodstream,

together

with

_only

a small amount of

_{triiodothyronine (T3) according}

to Lam et al.

_(1986).

The

_major

propor-tion of

_circulating

T3 is

_{produced by peripheral}

_{monodeiodination,}

which takes

_place

in the liver and in other tissues. Both T3 and IGF-I exert a

_negative

feed-back effect on GH secretion

_(Harvey,

_{1983; Buonomo et al.,}

_1987).

Effects of the sex-linked dwarf mutation

during growth

State of

_knowledge

in 1976

According

to the review

by

Guillaume

_(1976),

dwarf chicks exhibited

_{hypothyroidism}

which could not be

entirely

held

_responsible

for the dwarfism. No

_{significant deficiency}

was found at the level of the

_{thyroid gland.}

The saturation status of the

_thyroid

hormone

binding

protein

was used as a test to measure

_{triidothyronine plasma}

level and dwarfs showed

higher

in vitro

uptake

of

T3 than the normal birds

_(Grandhi

and

Brown,

1975a).

However,

the authors

_suggested

a failure to utilize

thyroid

hormones

_(Grandhi

and

Brown,

1975b)

or a difference in the cellular

_transport

of T3 or T4

_(Grandhi

et

al.,

1975).

Treatment with iodinated

proteins

(protamone)

could not

_compensate

for the final

_growth

retardation of dwarfs. The avian

_growth

hormone was not

_purified

until 1974. No direct

immunoassay

of GH had been

_performed

on dwarf birds. Somatomedins were known but could

_only

be studied

_{by biological}

assay: serum from dwarf chicks seemed to be able to

enhance

_sulphation

in chick

_cartilage

as well as or even better than serum from normal chicks. The

_working

_hypothesis

was thus a defect in tissue

_sensitivity

to

_growth

factors. This was in

_agreement

with the observation that the difference between dwarf and

nor-mal birds remained after

_{hypophysectomy.}

These data have now been

_updated

_following

the use of new

_{technologies :}

radioim-munoassays for T4 and

T3,

GH

(homologous),

IGF-I

_{(heterologous),}

assay for

GH-recep-tors, in vitro

_techniques

for cell

_physiology.

It should be noted that many studies compare a dwarf to a normal strain.

However,

a more accurate and reliable evaluation of the

_gene’s

effect can be obtained from the

com-parison

between dwarf and normal full

sibs,

bom from

_heterozygous

sires and dwarf dams. A

_linkage

_{with the feather color gene,}

silver,

enables the distinction of dwarfs from normals to be made at the

_embryo

_stage

_(Huybrechts

et

_al.,

_1986).

Two

_types

of

_birds,

broilers or

_laying

_strains,

are _{also studied which may}

_explain

some of the differences bet-ween studies.

Recent

_{endocrinological}

studies on dwarf mutation

Thyroid

hormones

Descriptive study.

Circulating

levels of T3 and T4 did not differ between normal and dwarf

_embryos

from 18

days

of age to the time of

_{hatching (Huybrechts}

et

al.,

1986).

However,

an

_important

difference arose after

_hatching:

the T3 level exhibited a

_significant

increase in the normal chick that was not seen to the same extent in the dwarf. Dwarfs showed lower levels of T3 from the _age of 3 to 9

weeks,

but

slightly higher

levels of T4 from the _age of 1 to 7 weeks.

_Many

authors now _agree that sex-linked dwarf chicks have decreased

_circulating

levels of T3 but normal or

_slightly

increased levels of T4

(May

and

Marks, 1983;

Scanes et

al., 1983;

Stewart et

al., 1984;

Hoshino et

al., 1986;

Lauterio

et al.,

1986).

This has

_justified

the

_study

of

_peripheral

monodeiodination in dwarfs.

Peripheral

monodeiodination The

_pathways

_of

_thyroid

hormone deiodination and their

regulation

are well described in the rat

_by

in

vivo

and in vitro studies

_(Visser

et

al.,

1984).

tis-sues such as

liver,

_kidney

or

_{muscle; type}

II in the brain and

_pituitary;

_type

III in the brain and

_placenta.

In vitro studies have not been

developed

to the same extent in the chicken.

According

to Fekkes ef aL

_(1982),

in the rat the same liver enzyme is

_responsible

for the 5 and the 5’ deiodination

_(5-D

and

_{5’-D) leading respectively}

to reverse-T3

_(rT3),

_inactive,

or

T3,

active.

_Circulating

level of rT3 was found to be similar and very low in normal and dwarf

chicks,

_suggesting

a rather normal 5-D in dwarfs

_(Hoshino

et

a/.,

1986).

The

regu-lation of the 5’- or 5- site of deiodination is not

_fully

_understood,

but could be related to

intracellular

_{pH (Visser}

et

_al.,

_1979).

The

regeneration

of the enzyme in its active form

requires sulfhydryl

groups

(Visser

et

al.,

1976),

which were shown to be

_normally

_present

in the liver of dwarf birds

_(Decuypere,

_{unpublished data).}

The deiodination

activity

is

influenced

by

feeding

status and

_temperature:

_fasting

decreases it and

_refeeding

has a

stimulating

effect on 5’-D in the chicken

_(Decuypere

and

Kuhn,

1984).

This

_refeeding

effect could be related to insulin rather than to

_{glucose, according}

to an in vitro

_study

in the rat

(Sato

and

Robbins,

1981

). Cold

also increases the deiodination

activity (Rudas

and

Pethes,

1984, 1986).

Ovine

prolactin,

GH and also TRH were able to stimulate

_peripheral

monodeiodination in the chick

_embryo

and the adult chicken but not in the

_growing

chick

_(Kuhn

et

al., 1983,

1988).

Indeed,

the

_sharp

increase in T3

_following

the

_perforation

of the inner shell

mem-brane

just

before

_hatching

is due to a

_higher

conversion rate of T4 to T3

_(Decuypere

et

al.,

_1982).

This increase in T3 could be induced as

_early

as _{the age} of 18

_days

_by

an

injection

of ovine

_prolactin

or

_growth

_hormone,

but such a stimulation could not be obser-ved in dwarf

_{embryos (Kuhn}

et al.,

1986).

This lack of _{T3 response in dwarfs} was

explai-ned

_by

a malfunction at the level of the

_peripheral

monodeiodination.

_Similarly,

a lower in vitro

_activity

of the liver 5’-D was found in chicks of a dwarf

_Leghorn

strain

_compared

to a normal

_Leghorn

strain

_(Scanes

_{ef al.,}

_1983).

_However,

this

could

not be confirmed in

comparing

dwarf and normal sibs within a strain of

_brown-egg

_layers

at

4,

8 and 12

weeks of age

(Decuypere

et al.,

1986).

The in vitro assay was

_performed

on the superna-tant fraction obtained after

_{centrifugation}

of

_homogenized

liver

tissue,

and in this

_system

the enzyme was shown to be

_present

and to work

_normally.

_However,

its

_activity

appea-red much appea-reduced in its cellular environment

_(i.e.,

in

_vivo).

This could

_suggest

a defect in the cellular environment of the enzyme and may in

particular

raise the

_question

of the

availability

of T4 as a substrate to the enzyme. The T4

_uptake

into

_hepatic

cell has been shown to

_depend

on both a

_passive

_{diffusion process and} an active mechanism

_requiring

ATP.

_Furthermore,

in rats, the

_{specific hepatic}

_receptor

_{sites for T4 may be different from} the sites for T3

_(Gharbi

and

Torresani, 1979;

Krenning

et al.,

1978, 1981

). A

defect at this level would be in

_agreement

with a normal in vitro but reduced in vivo

deiodination,

as

found in dwarfs. It is

_interesting

to note that the

_{hypothyroidism}

of dwarfs is not related to

thyroid, but

to the

_peripheral

monodeiodination

_activity

that was not studied until

_recently

in relation to fowl dwarfism.

Stimulation

_experiments.

The T4 release could be stimulated

_by

TRH both in normal and dwarf

_chicks,

with a

_tendency

to show a

_delayed

increase of T4 in dwarfs

(Michels

et

al., 1986;

Hoshino et

al.,

1986). Depending

on _{age, TRH had} no effect on T3 level in young chicks and elicited a

_slight

increase of T3 at 11 _{weeks of age in normal birds}

_only,

again suggesting

a lower

_ability

of dwards to convert T4 into T3.

Supplementation

experiments. Dietary

T4 did not

_{significantly change}

the

_growth

curve

of dwarf chicks from 1 to 9 weeks _{of age;}

however,

a

_positive

effect on

_{body weight}

could

ppm) significantly

depressed

growth (Leung

et

al.,

1984a).

The T4 treatment

_slightly

increased the T3

_plasma

level of

dwarfs,

but to a much lesser extent than the stimulation found in normals. A T3

_dietary

treatment could stimulate

_growth

of dwarf chicks at a dose of 0.1 or _{0.3 ppm in} some but not in all studies

_(Leung

et

al., 1984;

Marsh et

_{al., 1984b;}

Bowen

et al., 1987;

Tixier-Boichard et al., submitted for

_{publication).}

A

_genotype

x treat-ment interaction was observed when normal chicks became

hyperthyroid

and had a

reduced

_growth

rate,

while

_growth

of dwarf chicks was

_improved

or not affected.

_Higher

doses

_{(1 -10 ppm)}

had a

_negative

effect due to an induced

_hyperthyroid

status in dwarfs

(Leung

et al., 1984a;

Scanes

et al., 1986;

Tixier

et al.,

1986).

However,

consistent

meta-bolic effects were

observed,

suggesting

a normal

_sensitivity

of dwarf tissues to

T3,

such

as an increased rectal

_temperature,

a deterioration of feed

_efficiency,

a decrease in abdominal fat content, a decrease in GH level as a

_negative

feedback effect

_(Leung

et al., 1984a;

Tixier

et al.,

1986).

This last

_point

_justified

the simultaneous administration

of GH in some

_experiments

of T3 or T4

_{supplementation (Marsh}

et

al., 1984a;

Bowen

et

aL,

1987).

Together

with

_T3,

GH succeeded in

_{stimulating growth}

of dwarf

chicks,

though

treated chicks

did

not

_fully

reach the

body

weight

of the control normal chicks.

Growth hormone and insulin-like

_growth

factor I

Descriptive

studies. The

_plasma

levels of GH and IGF-I did not differ between

geno-types

at the end of the

_embryonic

life

_(Huybrechts

et

al.,

1987)

as had

_already

been

observed in

thyroid

hormones.

Indeed,

no difference in

_{body weight}

_appears at

_hatching

between

_genotypes.

Dwarf chicks showed a normal or even

_{higher circulating}

level of GH from the age of 3 to 9 weeks. The literature data show either a

similar

or an increased

GH

plasma

level in dwarfs as

_compared

to normals

_(Hoshino

et al., 1982;

Scanes

et al.,

1983;

Stewart et

_{al., 1984;}

Lauterio et

_{al., 1986;}

Bowen et

al.,

1987).

However,

IGF-I

plasma

level was

_always

found to be reduced

_by

about 50%

_(Hoshino

et

al., 1982;

Huy-brechts

et al., 1985a;

Bowen

et al.,

1987).

These results

clearly

show a deficient

synthe-sis of IGF-I in dwarf

birds,

which constitutes new information

_regarding

the mutation. The

growth

hormone of dwarf chicks is

immunoreactive,

although

its

_bioactivity

has not

_yet

been

_proved,

but

_{electrophoretic}

studies

_suggested

a

_{good similarity}

between the

circula-ting growth

hormone found in dwarf and that found in normal birds

_(Hoshino

and

Suzuki,

1982).

Stimulation studies. The increase in GH

_plasma

level after

_injection

of hGRF or TRH was of

_greater

_magnitude

and lasted

_longer

in dwarf than in normal birds

_(Hoshino

_{et al.,}

1984;

Huybrechts

et al.,

1985b).

TRH administration could also increase the GH

_plasma

level in adult dwarf

hens,

but had no effect in normal hens

_(Harvey

_{et al., 1984;}

Scanes

et

_al.,

_1986).

These results

_suggested

that dwarfs showed a

_{higher sensitivity}

to

_growth

hormone

_releasing

factors or a

_greater

_pool

of GH because of their lower T3

_circulating

level,

which

_normally

exerts an

_inhibitory

action on GH

synthesis

and release in birds.

Surgical

or chemical

_{thyroidectomy}

has been shown to

_promote

TRH-induced GH

secre-tion in adult birds

_(Harvey

et

_al.,

_1988).

Furthermore,

the

_dietary

administration of T3 to

dwarf birds could decrease a TRH-induced GH secretion

_(Scanes

etal., 1986;

Tixier-Boi-chard et

al.,

submitted for

_{publication).}

The slower decrease of GH

_circulating

level could also indicate a slower rate of

_peripheral

utilization of the hormone.

The stimulation of IGF-I

_synthesis

by

GH

_appeared

to take

place

in

_dwarfs,

but the

relative increase in IGF-I

_plasma

level was much smaller than in normal birds

GH

_receptors.

The low

_sensitivity

to

GH,

as

_recently

described

_by

_Huybrechts

et al.

(1988),

combined with the

_deficiency

of basal IGF-I

_production

in

dwarfs,

in

_spite

of

_high

levels of

_circulating

GH,

could

_suggest

a _{lack of response of the}

_hepatic

cell to GH.

Indeed,

hepatic binding

of avian GH has been shown to be

markedly

reduced or almost absent in dwarf birds of 2 different strains

_{(F.C. Leung}

et

al.,

1987).

These results also

suggested

a role for the

_{genetic background}

since no

_GH-binding

at all was noticed in dwarfs from a

_laying

strain

_(Leghorn),

while a few dwarf birds from a broiler strain

exhibi-ted a low

_binding

level. The authors found a

_higher

correlation between

growth

rate and

hepatic

GH

_binding

than between

growth

rate and GH

_plasma

level.

However,

the

possi-bility

of a

_{down-regulation}

of the GH

_receptors

_by

the hormone itself cannot

_yet

be ruled

out in dwarfs and needs further

_{investigation.}

IGF

_receptors.

The

_receptors

for IGF-I have not

_yet

been

_directly

studied in dwarf birds. In vitro studies did not show any

systematic

difference in the basal level of

carti-lage

sulfation

_activity

or DNA

_synthesis

between dwarfs and normals as shown

_by

a bet-ween-strain

_comparison

at 7 _{weeks of age}

_(Hoshino

et al.,

1982)

and

_by

a within-strain

comparison

from 17

_days

of incubation until 5 weeks of _age

_{(Charrier, unpublished}

results;

Figure 2).

The

addition

of normal chicken serum at the concentrations of 2.5 and 5% in the incubation medium of

_cartilage

tended to stimulate the

_cartilage

_activity

in dwarfs and in

normals,

but

_only

in a few cases so that no

_significant

differences could be established between

_genotypes

_(Hoshino

_{et al., 1982;}

_Figure

_3).

_However,

the addition of 20% of normal chicken serum to the incubation medium could stimulate the sulfation

only

be enhanced

_by

the addition of 20% normal serum at 7 and 13

weeks,

with no

signi-ficant differences between

_genotypes.

These results showed a normal

_activity

of

_cartilage

in

dwarfs,

with a somewhat

_higher

sensitivity

to serum

_growth

factors,

but

_they

should be confirmed on a

_{larger sample}

of

animals,

with the use of defined

_growth

factors in the culture medium. It must also be

emphasized

that these data were obtained from

_laying

strains and that the situation

might

differ in

_heavy

strains.

Corticosteroids

Dwarf birds showed a lower

_circulating

level of corticosterone from 1 to 6 weeks of age and no further difference at 12 _{weeks of age.}

Furthermore,

the

_plasma

corticosterone response to an acute heat stress at 12 weeks of _age was lower in dwarfs than in

nor-mals. In vitro studies also

_suggested

a decreased cellular

_sensitivity

to ACTH of the adrenal

_gland

in dwarfs as

_compared

to a normal strain

_(Carsia

and

Weber,

1986).

These results are not

_particularly

related to the decreased

_growth

in

dwarfs,

but the behavioral

aspects

and response to stress are

_interesting

to consider.

However,

age should be taken into account since adult dwarf hens were shown to have

higher

corticosterone

plasma

levels,

in a fed state as well as

_during

starvation

_(Rombauts

_{et al.,}

_1983).

Insulin

Dwarf birds showed rather lower levels of insulin after

_refeeding

or a

_glucose

load

(Simon,

1983).

However,

glucose plasma

level was not different from the control after

genotypes,

but it lasted

_{slightly longer}

in dwarfs before

returning

to the same basal

level,

90 minutes after the initial load. These results

_suggested

an increased

_sensitivity

to insu-lin action that could be related to an inefficient

_growth

hormone.

Summary

of the effects of the dwarf _gene

_{during growth}

The modifications induced

_by

_{the sex-linked dwarf gene}

_{during growth}

are

_presented

in

Table I. The Mendelian inheritance of dwarfism

suggests

the search for a

_single

abnor-mality

which could

_explain

the different effects observed. On the one

_hand,

dwarfs have

a reduced

_synthesis

_{of T3 in the presence of} an increased T4

level,

which could be rela-ted to lower in vitro monodeiodination

_activity,

in

_spite

of the presence of the enzyme; on

the other

hand,

the

_synthesis

of IGF-I is

_depressed

and the GH

_{hepatic binding activity}

is reduced or almost

absent,

in the presence of a normal or even increased GH

_circulating

level. The

_primary

effect of the gene may thus be at an interaction

_point

between the GH and IGF-I axis and the

thyroid

hormones.

In normal

_birds,

chemical or

_surgical

_{thyroidectomy}

decreased the

_circulating

level of

IGF-I but increased the GH level

_(Decuypere

et al.,

1987).

Chemical

_{thyroidectomy}

was

achieved

by

the administration of methimazole

(MMI);

in that case

_growth

of normal chicks was

_{greatly impaired}

because not

only thyroid

hormones but also IGF-I levels

were decreased. This could

_suggest

that a normal

_thyroid

function is

_required

for a pro-per

synthesis

of IGF-I. Treatment of dwarf birds

_by

T3 had a

_strong

_negative

effect on

either

_passive

or active

_(Krenning

et

al.,

1982).

T4 administration to dwarf chicks had

some

_{physiological}

effects on

_{body weight,}

on T3 and GH

_plasma

levels,

suggesting

that these birds were not resistant to the hormone. The T4 metabolism

_abnormality

in dwarfs

does not seen to be an all-or-none trait. A normal

_passive

_transport

_may

_explain

the effects observed with T4

_{administration,}

but the low level of T4 to T3 conversion and its lack of response to an

_injection

of GH or

_prolactin

in the

_embryo

_may

_imply

a defect in a

regulated

active

_transport.

The absence of the

_receptor

cannot be ruled out, but the defect could be due to a

_change

in

_receptor

_affinity,

or to a modified

_regulation

process at a level which has

_yet

to be determined.

Reciprocally,

GH has been shown to stimulate the T4 to T3 conversion in the normal

embryo,

with no effect in the dwarf. In the

chick,

this GH effect is no

_longer

_seen, but the conversion of T4 into T3 occurs at a much

_higher

rate than in the

_embryo.

Is it

_possible

that a

_hypothetical

_positive

effect of GH on the T4 to T3 deiodination

_might

no

_longer

be enhanced

by

exogenous hormone at that

_{age ?}

_Considering

that

_growth

_{hormone may} be

_required

for a normal

_peripheral

deiodination of T4 into

T3,

a GH

_receptor

defect could

then be seen as a sufficient cause of both deficiencies in IGF-I and T3

_production.

The

injection

of GH into dwarfs of a

_brown-egg

_type

_layer

strain induced

_only

a small increase in the

_circulating

level of IGF-I

_(Huybrechts

et al.,

1988).

This small effect alone could be

seen as a sufficient reason to exclude the total absence of the

_receptor

in dwarfs.

How-ever, the results obtained in the

_Leghorn

strain

_by

FC.

_Leung

etal.

_{(1987) suggested}

that the GH

_receptor

could be

_totally

_lacking

in dwarfs or _{have lost any}

_{binding affinity}

for the

hormone,

but the results were not as clear-cut in the broiler strain. Further

_experiments

are

_required

to test the

_hypothesis

of an abnormal GH

receptor

in dwarfs. An

_interesting

stage

to

_study

would be the end of the incubation

_period

where the normal increase of T3

production

does not take

place

to the same extent in dwarfs and cannot be stimulated

_by

GH as in normals.

The

_target

tissues such as

_cartilage

or muscle have not been

_extensively

studied in dwarfs. Normal

_cartilage

_{may be able} to

_produce

its own

_growth

_factors,

in an autocrine

manner

_(Burch

et al.,

_1986),

so that it would not be

_{entirely dependent}

on the

_circulating

IGF-1.

However,

the

_circulating

IGF-I can still

_{play a}

_role,

as shown

_by

the effects of

exo-genous IGF-I on

_growth

of miniature

_{poodles (Guier}

et al.,

1987)

and of

hypophysecto-mized rats

(Schoenle

et al.,

1982).

In addition to in vitro

studies,

the simultaneous

com-pensation

for both T3 and IGF-I deficiencies in dwarfs would

_permit

an in vivo evaluation of the

_{growth potential}

of

_peripheral

tissues.

Relationships

between the effects _{of the gene}

_{during growth}

and its effects in the

laying

hen will be considered below.

Effects of the gene

during

the

_laying

_period

Descriptive

studies

The sex-linked dwarf _gene reduces

_{vitellogenesis}

and ovulation rate

_(Guillaume,

1976;

on the basal

plasma

levels of

_{1 7}

_f

_i.estradiol,

which would be

increased,

and of

progeste-rone, which would be decreased

(Tixier-Boichard

and

Rombauts,

1988). Consequently,

the ratio of estradiol to

_progesterone

would be

_higher

in dwarfs. This result was most

noticeable after the

_laying

peak

whereas no trend

appeared

earlier. The

_relationship

of this

_change

in the

_circulating

levels of sex steroids to the other

_{endocrinological}

effects of the mutation remains to be elucidated. It could also be a _{direct consequence of the}

redu-ced number of

_fast-growing

ovarian follicles in the dwarf hen.

Furthermore,

no

_significant

change

was observed in the

_luteinizing

hormone

_plasma

level around the onset of

_laying,

which could indicate a

_peripheral

defect;

but the ovarian

_sensitivity

to

_pituitary

factors has

not been studied in the dwarf hen.

So far as

_{vitellogenesis}

is

concerned,

the dwarf

_Leghorn

hen

_appeared

to be more

dependent

on the diet for the

_lipid

_composition

of the

yolk (Demarne

et

al., 1984;

Bur-ghelle-Mayeur,

1988).

The in vitro

lipolytic

and

_lipogenic

activities of the

adipose

tissue

were

_{significantly}

lower in dwarf

_Leghorn

hens while

hepatic

lipogenic

activity

was not

changed (Burghelle-Mayeur, 1988).

Adult dwarf hens have much less abdominal fat than their normal sisters

(Merat

and

Ricard,

1974),

but the

opposite

is observed at a _young

age where the dwarf chicks are fatter

_(Stewart

et

al.,

1984).

The effect _{of the dwarf gene}

on

_lipid

metabolism

_during

_growth

is not

yet

clearly explained:

lipogenesis

was found to

be decreased in a dwarf line at 4 weeks of age

(Rosebrough

et

al.,

1986),

but

contradic-tory

results were obtained

_depending

on the

_{genetic background}

where the gene was

introduced

_(Calabotta

et al.,

1983).

Results seem to be more consistent

_regarding

lipoly-tic

_activity

which

appeared

to be decreased in dwarf chicks

(Guillaume,

1976;

Calabotta

et

_al.,

_1983).

The balance between

lipogenesis

and

_lipolysis

may differ between the

growing

and the

_{laying periods,}

but a constant feature in dwarfs seems to be a reduced

ability

to mobilize their

_lipid

deposits.

This decreased

_lipolytic

activity

may be related on

the one hand to the decreased T3

level,

since T3 is

_lipolytic

in the chicken

(Decuypere

et

_al.,

_1987),

and on the other hand to a deficient

_growth

hormone or to a defect of the

GH

_receptor

since

_growth

hormone has been shown to be involved in the

_lipid

metabo-lism of the

chicken,

with either a

_{lipolytic activity}

in vivo

_(Campbell

and

Scanes,

1985)

and in vitro

(Hall

et

_al.,

_1987),

or an

antilipolytic

action in vitro

_{consisting.of}

a short-term

inhibition of the

_{glucagon-induced}

lipolysis

in the

_adipose

tissue

_(Campbell

and

Scanes,

1987).

The role of

_growth

hormone in the

_regulation

of

_lipolysis

could thus be

_interesting

to

_study

in the dwarf hen.

Consequences

of the hormonal deficiencies associated with dwarfism

_during

the

_laying

period

Lower T3

_production

T4

_plasma

levels were increased

_during

the

_{laying period}

in dwarf

_Leghorn

hens as

compared

to normal

hens,

while the

_circulating

levels of T3 were rather

decreased,

this

pattern

being

very close to the situation described at an

_early

_age

_(Decuypere,

unpub-lished

_data).

In a

_{preliminary experiment,}

where a small

_sample

of dwarf hens received

0.1 _ppm T3 in the diet from 18 to 22 weeks of _{age, the}

_plasma

level of T3 was

_slightly

increased while the T4 level was

_decreased,

but no

_significant

effect could be noticed on

laying

traits

_{(Tixier-Boichard, unpublished}

results).

An inverse

_relationship

is

_usually

broo-diness

_(Sharp

and

Klandorf,

1985). Thyroid

hormones seem to have an

_inhibitory

action

on the secretion of

_luteinizing

hormone in the fowl

_(Harvey

et

al.,

1983). They

also

appeared

to have an

_inhibitory

effect on the

_{estrogen-induced vitellogenin}

_synthesis

in the chick

_embryo

liver

_(Elbrecht

and

Lazier,

1985).

However,

a minimal level of

thyroid

hormones may be necessary for a chicken to exhibit a normal response to an

_estrogen

challenge,

as shown in the obese strain of White

_Leghorn

_showing

inherited

hypothyroi-dism

_(Schjeide

et al.,

1986).

Sex-linked dwarf cockerels have a T3

_deficiency,

_but appea-red to be as sensitive as _{their normal half-brothers in response} to an

_injection

of

17p-estradiol

_{(5 mg/kg)}

at 7 _{weeks of age: the}

_plasma

level of

_{triglycerides}

reached a

maximum 28 h after the

_injection,

with a mean

_{(± SEM)}

of 5.88 ± 0.94

_g/l

in dwarfs as

compared

to 1.95 ± 0.72

_g/i

in untreated dwarfs and a mean of 7.19 t 0.28

_g/i

in normals

as

_compared

to 1.96 ± 0.58

_g/i

in untreated normals

_{(Tixier-Boichard, unpublished}

results).

The

changes

in

_thyroid

metabolism due to the dwarf _gene are not

_easily

related to the effects of the gene on

_laying

_traits,

_indicating

that the

_{relationships}

between

thyroid

hor-mones and steroid hormones

_{during reproduction}

should be further studied in the dwarf hen.

Lower

_{IGF-I production}

A

_possible

role of IGF-I in the

_reproductive

function has not

_yet

been

_investigated

in the chicken. In

_mammals,

some studies

_suggest

an IGF-I involvement in the

reproductive

function,

as for instance the

_synergism

with Follicle

_Stimulating

Hormone to induce

receptors

for

_Luteinizing

Hormone in cultured rat

_granulosa

cells

_(Adashi

et al.,

1985).

The

_plasma

level of IGF-I has been found to be reduced in dwarf

_pullets

_aged

15 and 18 8

weeks

_(Huybrechts

et al.,

1987)

but there are no data at an older _age.

Until now, the effects of the sex-linked dwarf gene on

_reproduction

and

_mainly

on

ovu-lation rate _appear

_complex

_{and may involve several}

_regulation

_pathways

that cannot be

easily

related to the marked modifications observed at a _{young age.}

Conclusions and further

_approaches

The studies of the sex-linked dwarf mutation in

_growing

chicks tend to favour the idea that the

_primary

_{effect of the gene is connected with the}

receptor

protein

for

_growth

hor-mone, from direct

_experimental

evidence,

or with the

_receptor

for

_T4,

from indirect

experi-mental

evidence.

Further

_{physiological investigations}

are

_needed,

_including

in vitro studies of T4

_uptake

and GH

_binding

in the liver as well as in vivo studies of GH and IGF-I effects in the dwarf. From another

_point

of

view,

molecular

_genetics

can offer a new tool to test the

_hypothesis

of an abnormal GH hormone or GH

_receptor

_{in the dwarf. The gene for chicken GH has} been cloned

_(Souza

et

_al.,

₁₉₈₄₎

and could be used as a

_probe

to detect a structural dif-ference of the GH _gene between dwarf and normal

chicks,

according

to the method of restriction

_fragment

_{length polymorphism.}

This difference should not be connected with the

_antigenic

_properties

of the

hormone,

since it is

_normally

_recognized

with the usual

pre-sented

_by

_Leung

et al.

_{(1984b) , showing}

no difference between the DNA from dwarf or

normal birds. The rabbit gene for the

growth

hormone

_receptor

has now been cloned

(D.W. Leung

et

al.,

1987)

and could

_provide

an

_interesting

tool to compare the dwarf to

the normal genome.

However,

the _{mutation may be connected with}

_only

a

_part

of the gene, the size of which is unknown. Since the entire

probe

may not be able to detect a

small

difference,

a modified or an

_{incomplete probe}

could then be useful. One can

spe-culate on a mutation of that gene that could affect the sequence involved in the

binding

of the hormone to its

_receptor.

If this sequence could be

identified,

it would

_help

in

establi-shing

a

_{&dquo;specialized&dquo; probe}

to be used in the dwarfs

suspected

of a

_GH-binding

defect.

These considerations are

_speculative,

but are aimed at

_showing

that both

physiology

and

molecular

_genetics

could now

_{play a}

_part

in

_elucidating

the nature of the dwarf mutation.

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