The characteristics of the melatonin secretory rhythm are not modified by the stage of pregnancy in ewes

Original

article

The characteristics of the melatonin

_secretory

_rhythm

are

not

modified

_by

the

_stage

_{of pregnancy in}

ewes

LA

_Zarazaga,

B

_Malpaux

P

Chemineau

Neuroendocrinologie sexuelle, physiologie de la _{reproduction,} Inra, 37380 _Nouzilly, France

(Received 4 _September 1996; accepted 23 October ₁₉₉₆₎

Summary ―

An

_experiment

was

_designed

to

_study

if,

in the same

_animals,

characteristics of the

_plasma

melatonin

_rhythm

_vary

_during

_{pregnancy in} ewes. Thirteen Ile-de-France ewes were maintained in natural

_photoperiod

and measurements of the characteristics of the

_rhythm

of melatonin secretion were determined

_during

one estrous

_cycle

and then

_during

_pregnancy.

Duration and mean

_plasma

concentrations of the nocturnal melatonin elevation were measured

at four

_stages

_{of pregnancy. Melatonin concentrations}

_during

the elevation were not

_{significantly}

affected

_by

the

_stage

of the estrous

_cycle

_{(mean ±}

SEM: 227.8 ±

34.9,

263.2 ± 24.2 and 232.4 ± 27.1

_pg/mL

for follicular

_phase,

_early

luteal

_phase

and late luteal

_phase,

_{respectively),}

or

_by

the

stage

of pregnancy

(292.8

±

22.2, 268.7

±

_{21.7, 267.4}

± 27.9 and 258.3 ± 23.5

_pg/mL

for

_first,

second,

third and fourth month of pregnancy,

respectively).

A

_strong

individual effect was

detected

_(P

<

_0.01)

for melatonin concentrations

_during

elevation

_(range:

119.2 ± 11.8 to

396.6 ± 26.0

_pg/mL

of

_plasma).

A

_{highly significant}

correlation coefficient was observed within individuals for

_night

melatonin concentrations between the different

_{physiological}

stages.

It was concluded that melatonin secretion is unaffected

_by

the

_stage

_{of pregnancy.}

melatonin / _{pregnancy / secretion} / ewe

Résumé &horbar; Les

_{caractéristiques}

du

_rythme

de sécrétion de mélatonine ne sont _pas

modi-fiées

_pendant

la

_gestation

chez la brebis. Les

_{caractéristiques}

du

_rythme

de sécrétion de la méla-tonine ont été étudiées

_{pendant la gestation}

en

_répétant

les mesures sur les mêmes animaux. Treize

brebis,

soumises à une

_{photopériode}

_naturelle,

ont été utilisées et les

_{caractéristiques}

du

_rythme

de sécrétion de la mélatonine ont été mesurées

_pendant

un

_cycle

oestrien et

_pendant

la

_gestation.

La durée de sécrétion et _{les concentrations moyennes de mélatonine}

_pendant

l’élévation noc-turne ont été étudiées à

_quatre

moments différents de la

_gestation.

Les concentrations

noc-*

Correspondence and _reprints

turnes de mélatonine ne sont _{pas différentes} entre les trois stades du

_cycle

étudiés

_(moyenne

±

SEM ; 277,8

±

34,9 ; 263,2

±

_24,2

et

232,4

±

27,1

pg/mL

pour la

phase

folliculaire,

le début et

la fin de la

_phase

lutéale,

respectivement),

ou les

_quatre

stades de

_gestation

étudiés

(292,8

±

22,2 ;

268,7

±

_{21,7 ; 267,4}

±

27,9

et

258,3

±

23,5

_pg/mL

pour le

premier,

deuxième,

troisième et

quatrième

mois de

_{gestation, respectivement).}

Une variabilité interindividuelle très

_importante

a été observée

_(p

<

_0,01)

_{pour les concentrations de mélatonine}

_pendant

la nuit

(écart

de

119,2 ±

11,8

à

396,6

±

26,0

pg/mL

de

_plasma).

Un coefficient de corrélation très élevé intra-individus

a _{été observé pour les concentrations de mélatonine} entre les différents états

_{physiologiques.}

Cette étude

_indique

_{que la sécrétion de mélatonine n’est pas modifiée par la}

_gestation.

mélatonine /

_gestation

/ sécrétion / brebis

INTRODUCTION

Diurnal

_production

of the hormone melatonin

by

the

_{pineal gland}

mediates the effects of the environmental

_photoperiod

to

_regulate

seasonal

_cycles

of

_reproduction

in a

_variety

of

mammals

_(Bittman

et

_{al, 1983;}

Goldman and

Darrow, 1983;

Karsch et

al, 1984;

Hastings

et

al,

1985).

It is still

unclear, however,

which of the characteristics

_(amplitude,

duration or

phase

of

secretion)

of that

_rhythm

_conveys the

_{photoperiodic}

information to _the

repro-ductive axis. The

_{amplitude -}

the difference between melatonin concentrations

during

the

day

and the

_{night -}

has not received much consideration in

_sheep

because it does not

differ between

_long

and short

_days

and it

appears to _{be very variable between animals}

(Malpaux

et

al,

1987).

In contrast, the dura-tion of secredura-tion varies between

_long

and short

_days,

but whether it is the duration of

melatonin secretion per se, or _{its presence} at a

_{precise period}

of the

_night,

which conveys the

_{photoperiodic}

information to _the

repro-ductive axis has

yet

to be determined

_(Wayne

et al,

1988).

While the role of the diurnal

_rhythm

in

plasma

melatonin concentrations in the ovine foetus is

unknown,

it has been demonstrated that the

_reproductive

_{response of the}

new-born and young to the

_{postnatal photoperiod}

is

_markedly

affected

_by

the

_photoperiod

experienced by

the mother

_during

_pregnancy

(Horton,

1984;

Stetson et

al, 1986;

Weaver

and

_{Reppert, 1986).}

This information may

be used

_jointly

with the

_{postnatal photoperiod}

to induce

_seasonally

_appropriate

reproduc-tive and behavioral responses in the young lamb

_(Zemdegs

et

al, 1987;

Ebling

et

al,

1989;

Foster et

al,

1989).

Whether melatonin

concentrations _vary

_during

pregnancy is not

clear. Some studies

_suggest

an increase in melatonin concentrations

_during

the last

trimester of pregnancy in humans

(Birau

et

al,

1984;

Kiveld, 1991).

In

_sheep,

an absence

of normal melatonin

_{rhythm during}

preg-nancy was

_suggested

_{by Kennaway}

et al

(1981),

but other authors did not

_report

_any

difference in melatonin concentrations

between two different time

_periods

of the last third of pregnancy

(Yellon

and

_Longo,

1987;

Zemdegs

et al,

1987).

Important

characteristics of melatonin secretion are its

_high

inter-individual

vari-ability (Malpaux

et

al,

1987)

as well as its

_high

repeatability

(Chemineau

et

_{al, 1996).}

It is therefore critical to

_study

the effect of

poten-tial

_regulatory

factors of melatonin

concen-trations on the same individuals.

_Recently,

we

have shown that melatonin secretion is not

modified

_by

steroid administration at

concen-trations

_typical

of the estrous

_cycle

nor

_by

the

stage

of the estrous

_{cycle during}

the seasonal

anestrous

_(Zarazaga

et

al,

1996).

The

objec-tive of this work was thus to determine whether melatonin secretion characteristics

_change

dur-ing

pregnancy

by

assessing

its secretion at

to that found at three characteristic times of the estrous

_cycle

of the same animals.

MATERIALS AND METHODS

Thirteen adult Ile-de-France ewes

_aged

2 to

6 years were studied.

_During

the entire

experi-ment, the ewes were fed

_daily

with

_hay,

straw

and corn, and had free access to water and min-eral licks.

_They

were maintained outdoors at

the INRA research center of

_Nouzilly,

France

(48°N)

during

the

_{study. They}

were isolated

from the rams and treated for 12

_days

with an

intravaginal

pessary

(30

mg acetate of

fluoro-gestone,

FGA; Intervet-AKZO,

Boxmeer, the

Netherlands),

and

_injected

_{with 400 IU of}

preg-nant mare serum

_{gonadotrophin}

_(PMSG;

Inter-vet-AKZO)

at the time of sponge withdrawal

to induce

_synchronized

estrus and ovulation. Withdrawal of the pessary was considered as

the first

_day

of the estrous

_cycle

( 15

Septem-ber =

_day

_0,

_dO).

In order to obtain a

measure-ment of melatonin

_plasma

concentrations before the establishment of pregnancy

(control values),

melatonin concentrations were measured at three

stages of the estrous

_{cycle preceding}

fertilization:

dl, follicular

_phase;

d5,

early

luteal

_phase

and

d14, late luteal

_phase.

The ewes were

subse-quently

mated with Ile-de-France males at the

next natural estrous

_cycle

and melatonin

con-centrations were measured at four different times

during

pregnancy: the first

(mean

± SEM: 29 ±

0.3

_days

of pregnancy, range: 27-31

days),

sec-ond

(57

± 0.3

_days

of

pregnancy),

third

(90

±

0.3

_days

of

_pregnancy)

and fourth

₍₁₁₉

± 0.3

days

of

_pregnancy)

_{months of pregnancy. The}

stage of pregnancy was calculated from the date of

_{mating. Figure}

1 shows the

_{general design}

of the

_experiment.

Sunset and sunrise hours were official hours

provided by

a

_{meteorological}

station located at

about 10 km from the

_experimental

location.

Blood was

_sampled

for melatonin

inter-vals,

starting

and

_ending

2 h before and after sunrise and sunset,

respectively. During

the hours of _darkness,

_samples

were collected under dim

red

_light

_(<

3

lux)

avoiding

any direct illumina-tion of the animal’s eyes. Plasma was

immedi-ately separated by centrifugation

and stored at

- 20 °C until assay.

In addition,

during

the estrous

_cycle

ovarian

activity

was measured

_{by obtaining plasma}

sam-ples

for progesterone determination from dO to

d19, and ovulation rate

_(OR)

was observed on

dl2

_{by laparoscopy using}

the

_technique

described

_by

Oldham and

_Lindsay

( 1980).

Hormonal

_analysis

Plasma melatonin concentrations were

_assayed

by radioimmunoassay using

the

_technique

described

_by

Fraser et at

(1983)

with

_antibody

first raised

_by

Tillet et at

_(1986),

in

_duplicate

aliquots

of 100

_pL

of blood

_plasma.

The

sensi-tivity

of the assay was 4 ± 0

_pg/mL

(two

assays)

(melatonin

Fluka-63610).

The coefficient of variation was estimated

_{by assaying}

three

_plasma

pools

(low,

medium and

_high

concentrations of

melatonin)

in

_duplicate

_{for every 100 unknown}

samples.

The

_intra-assay

coefficient of varia-tion was 10.6%. The

_inter-assay

coefficient of variation was 6.3%

_(two

_assays).

Plasma _progesterone concentrations were

assayed by radioimmunoassay using

the

tech-nique

described

_by

Saumande et al

(1985).

The

sensitivity

of assay was 0.125

_{ng/mL. Intra-assay}

coefficient of variation was 17.7%. All

_samples

were

_assayed

in the same _assay.

Data

_analysis

For each _stage of the estrous

_cycle

_and

preg-nancy and for each animal, a melatonin elevation

was defined

_according

to

_Malpaux

et a1

(1987).

Two characteristics of the melatonin elevation

were

_compared:

the duration

_(defined

as the time

elapsed

from the first

_sample

of the elevation to

the first

_sample

after the end of the

_elevation)

and the mean melatonin concentration

_during

the elevation.

An

_analysis

of variance with

_repeated

fac-tors was used to test the stage of the estrous

cycle

and pregnancy

(seven

stages in

_total)

and individual effects on mean melatonin

concen-trations and duration of the elevation

(Super-ANOVA,

Abacus

_{Concepts, Berkeley,}

CA, USA).

The correlation coefficient between

proges-terone levels and melatonin concentrations at

each _stage of the estrous

_cycle

was calculated. The

_{repeatability}

of mean melatonin

concentra-tions and duration of the elevation from

_night

to

_night

was estimated

_by

the correlation coef-ficient.

RESULTS

The _patterns of mean

_plasma

melatonin

con-centrations

_during

each

_stage

of the estrous

cycle

and

_during

each of the four studied

stages

of pregnancy are

_presented

in

_figure

2. All ewes

_presented

a clear

_rhythm

in their

plasma

melatonin

concentrations,

with a

_rapid

increase after the onset of

darkness,

a

_rapid

decrease after sunrise and undetectable

val-ues

_during

the

_day.

No

_significant

effect of the different

phys-iological

stages

was detected on the mela-tonin concentrations

_during

the

_period

of ele-vation

_(mean

± SEM: 277.8 ±

_34.9,

263.2 ±

24.2 and 232.4 ± 27.1

_pg/mL

for follicular

phase, early

luteal

_phase

and late luteal

_phase,

respectively,

and 292.8 ±

22.2,

268.7 ±

21.7,

267.4 ± 27.9 and 258.3 ± 23.5

_pg/mL

for the

first,

second,

third and fourth month of preg-nancy,

respectively).

A

_highly

_significant

indi-vidual effect was detected for this

_parameter

during

the

_experiment

(P

<

0.01)

(range:

119.2 ± 11.8 to 396.6 ± 26.0

_pg/mL).

The correlation coefficients for mean

_night

melatonin concentrations between the _stages

A

_significant

effect of the

_photoperiod

(ie,

stage of the

_experiment)

on the duration of melatonin elevation was observed

(mean

±

SEM:

11.0±0.1,

10.9±0.1 and 11.9±0.2h h for follicular

_phase,

_early

luteal

_phase

and late luteal

_{phase, respectively,}

and 13.7 t

_0.2,

13.5 ±

_0.2,

13.8 ± 0.2 and 12.8 ± 0.2 h for

first, second,

third and fourth

months,

respec-tively,

P <

_0.05).

No individual effect was

detected for this

parameter

(range:

12.0 ± 0.5

to 13.0 ± 0.6

h).

Only

the correlation coeffi-cient between the follicular and

_early

luteal

phase

was

_{significantly}

different from zero:

0.83

_{(P < 0.01}

).

Figure

3 shows

changes

in

_plasma

pro-gesterone

concentrations

during

the estrous

cycle

with

_respect

to the time of determination of melatonin levels. Plasma

progesterone

con-centration was low

_during

the follicular

_phase

(0.13

± 0.00

_ng/mL),

increased

_during

the

early

luteal

_{phase (0.31}

± 0.05

_ng/mL)

and

was

_high

_during

the late luteal

_{phase (3.91}

±

0.36

_ng/mL).

Correlation coefficients between

progesterone

levels

_during

the estrous

_cycle

and melatonin concentrations

_during

the

ele-vation at the different

_stages

of the estrous

cycle

were low and not

_{significantly}

differ-ent from zero.

DISCUSSION

These results demonstrate that melatonin

con-centrations studied

_by

_repeating

measurements at seven different time

_periods

in the same

animals are not modified

_by

the

_{physiological}

stage of the

animal;

melatonin characteristics

are not modified

_during

the estrous

_cycle

nor

during

pregnancy.

The results

_presented

here

_concerning

the

effect of the _stage of the estrous

_{cycle during}

the

_breeding

season on melatonin

character-istics,

confirm our

_previous

observations on

the absence of

_significant

effects of adminis-tration of steroids

(estradiol

and

_{progesterone)}

at concentrations

_typical

of the estrous

_cycle

and the absence of a

_significant

effect of the

stage of estrous

_{cycle during}

seasonal ane-strous

_(Zarazaga

et

al,

1996).

We can

con-clude that melatonin characteristics are not

modified

_by

the _stage of the estrous

_cycle

in

ewes,

irrespective

of the season

_(spring

and

autumn).

Concerning

pregnancy, there is some evi-dence in the literature for increased function

of the

_{pineal gland compared}

_with

nonpreg-nant

animals,

which could

_suggest

an increase

in melatonin concentrations in

_peripheral

blood. For

_{example, morphological}

and his-tochemical

_changes

of the

_pineal

_gland

have been described

_during

pregnancy: Pevet and Smith

(1975)

showed an increase in the

quan-tity

of

_Golgi

_apparatuses

in the mole and Nesic and Kadic

(1979)

showed

_changes

in the

nuclear volume of

_pinealocytes

in the ewe

pineal gland.

However,

Huang

and Everitt

(1965)

showed a decrease in

_{pineal weight}

of rats with ten or more fetuses.

Concerning

the melatonin secretion

_during

pregnancy, Birau et al

(1984)

and Kiveld

(1991),

reported

an increase in melatonin

con-centrations in

humans,

in the last trimester of pregnancy

compared

to the first and second trimester and Zimmermann et al

(1989)

observed an increase in melatonin

found no normal

_nightime

rise in the

_plasma

melatonin levels

_(Kennaway

et

al,

1981).

In the

_present

_study,

all ewes had a clear diurnal

rhythm

in

_plasma

melatonin concentrations

during

pregnancy, as is

_generally

observed

dur-ing

other

_{physiological}

_stages.

In contrast to

the above observations in

humans,

Zemdegs

et al

(1987)

and Yellon and

_{Longo (1987)}

did

not find differences in melatonin

concentra-tions between two different times of the last trimester

_{of pregnancy}

(120

vs 135 and 114 vs

142

_days

_{of pregnancy,}

_{respectively)}

in

_sheep.

Our

_study

extends these observations

_by

show-ing

that until d120 of pregnancy melatonin concentrations do not _vary.

Melatonin secretion shows a very

high

inter-individual

_{variability (Malpaux}

et

al,

1987).

It is

_possible

that the lack of consistent results on the effect of the

_stage

of estrous

cycle

or _pregnancy on melatonin

concentra-tions or

_pineal

characteristics could have

resulted from the inter-individual

_comparisons.

Therefore, in

the

_present

_experiment,

we

stud-ied the influence of

_{physiological}

conditions on

the melatonin characteristics

_by

_repeating

the

measurements in the same individuals. Under these

conditions,

we found that melatonin

con-centrations at three critical

_stages

of the estrous

cycle

and four

stages

of pregnancy were not

different. These results

_suggest

that melatonin secretion in the ewe is not influenced

_by

repro-ductive

_{physiological}

_stage.

Regarding

duration of melatonin

secretion,

it should be stated that the

_design

_{of the}

pre-sent

_experiment

was not

_fully

_adequate

to

monitor its variation

_during

pregnancy, as the

animals were maintained in open

barns,

and not in controlled

_{photoperiodic}

conditions. The animals were maintained outdoors on a

natural

_photoperiod

from

_September

to Febru-ary, a

_period

_during

which

_daylength

_changed

from about 12.5 to 10.0 h.

Therefore,

the effects of the

_{physiological}

conditions of the

animal are

_potentially

confused with effects of

changing daylength.

In

conclusion,

the overall results and those of our

_{previous study}

_(Zarazaga

et

al,

1996),

show that melatonin concentrations are not

modified

_during

the estrous

_cycle

and

demon-strate that the

_amplitude

of the

_rhythm

of melatonin secretion is not modified

_during

pregnancy in ewes.

Moreover,

the

_high

inter-individual effect detected on melatonin

con-centrations

suggest

that this

_parameter

is an

individual characteristic of each animal which

may have a

_strong

_genetic

basis.

ACKNOWLEDGMENTS

LZ was

_{supported by}

a

_postdoctoral

_grant from the

_Diputaci6n

General de

_Arag6n

and

_by

a

post-doctoral _grant from INRA-DSPA. The authors would like to thank A Daveau and F Maurice-Mandon for their advice on _{the technical}

pro-cedures. We also wish to thank P

_Lonergan

for revision of the

_{English manuscript.}

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