10 mm for I h except in the youngest age group. No significant sex difference in the. LH response to LHRH treatment was observed. The. LH response increased.
BIOLOGY
OF
REPRODUCTION
27, 556-561
(1982)
Response of Luteinizing Hormone and Follicle-Stimulating Hormone To Luteinizing Hormone Releasing Hormone in the Fetal Pig B. COLENBRANDER,’ N. PARVIZI Institute
A. A. MACDONALD,’ and D.F.M. VAN DE
of Animal
Husbandry Neustadt,
and
F. ELSAESSER, WIEL2
Animal
Behavior
F. R. G. and
Research
Institute Zeist,
for The
Animal
Husbandry
Netherlands
ABSTRACT
The responses of anesthetised luteinizing hormone releasing
fetal pigs (n95) hormone (LHRH)
and chronically
catheterized
fetal
pigs (n=10)
to
administration (2 pig/kg estimated fetal body studied at 55, 70, 85, 100, 106 (chronic) and 113 days. hormone (FSH) and luteinizing hormone (LH) were samples were taken from the umbilical artery (anesthetised fetuses) every 10 mm for I h except in the youngest age
weight) was investigated. Fetuses were Plasma concentrations of follicle-stimulating measured by radioimmunoassay. Blood fetuses) or carotid artery (catheterized group. No significant sex difference in the LH response to LHRH treatment was observed. The LH response increased with gestational age; average pretreatment plasma concentrations were below 1 .1 nglml. No response was observed at 55 days, and the highest response was seen at 113 days when plasma LH concentrations rose to 4.3 ± 0.7 (mean ± SEM) ng/ml 40 mm after treatment. Pretreatment plasma FSH concentrations at 55 days were 1.6 ± 0.1 nglml and gradually rose in males to 3.2 ± 0.4 nglml at 113 days, which was significantly lower than in females where concentrations averaged 8.1 ± 2.0 ng/ml. LHRH did not significantly affect FSH concentrations in males, while in females a gradually increasing response was observed; at 113 days plasma FSH was 12.5 ± 2.9 ng/ml 40 mm after treatment. The increase in response to LHRH with age of plasma LH concentrations in both sexes, and of plasma FSH concentrations in females indicates the maturation of the hypothalamo-pituitary system. INTRODUCTION
The
functional system
pituitary
development
in the
fetal
of
both
concentrations (LH) the
and serum
decline Several
secretion,
pituitary hormone by
the
1976; In
to
hypothalamus Mueller the
sheep
and
The
of
LHRH and a period
is probably
1980).
Since
a decline at the end
Sklar
due
gonadotrophic
testosterone secretion-at pig (Colenbrander et
the
feedback responsible
releasing
secretion
the
Grumbach, of
a!., 1976;
et
decline
by
a!.,
et
to a negative
by sex steroids and/or to a restraint of secretion in the hypothalamus due to maturational changes independent of gonadal sex steroids (Takagi et a!., 1977; Conte et a!.,
1981). human,
(Kaplan
This
hypothalamo-pituitary
LHRH
of
of
fetal
gestation is followed hormone secretion
feedback
et a!., be responsi-
sensitivity
the
gestation
1981).
(FSH) in period and
hormone
increased (Kaplan
et al.,
pig. hormone
high concentrations as increased autonomous
luteinizing
and
during
neonatal
hormone perinatal
increased
(LHRH)
changes
of
system in mid in gonadotrophic
hypothalamo-
(Colenbrander factors may
ble for the perinatally these hormones such pituitary
and
the
luteinizing
follicle-stimulating rise in the
subsequently 1977, 1982).
activity
activity of markedly
high
action for
male
in female
than
system. are believed gonadotrophic human Also,
1976).
androgen
concentrations in
female
fetuses
(Meusy-Dessolle, To study
the
556
the
are at
of
might activity Due
be of
to
this
to cause lower hormones in
higher
the
end
in
of
LHRH
in
male
gestation
et a!., 1980). changes
system effect
fetal male negative
fetuses (Kaplan and in the pig serum
1974; Ford maturational
hypothalamo-pituitary investigated
stimulate
in the 1979)-a
hormone in the
hypothalamo-pituitary androgens of
than
a!.,
by this the alteration
feedback, concentrations Grumbach,
Accepted April 19, 1982. Received December 21, 1981. ‘Present address; reprint requests: Dept. of Veterinary Anatomy, State University Utrecht, P. 0. Box 80157, 3508 TD Utrecht, The Netherlands. Research Institute for Animal Husbandry “Schoonoord”, P. 0. Box 501, 3700 AM Zeist, The Netherlands.
hormones least
the treatment
of
the
pig
we on
RESPONSE
concentrations fetal
of
pig
Samples well
during were
as
fetuses. with
from
plasma the
obtained
and
gestational
MATERIALS Animals German were used Acute thetised methomidate
and
during
chronically
We correlated sex
LH
second
Experimental
and
TO
FSH
in
half of gestation. fetal surgery
catheterized
stable
response
to treatment
the
LHRH
IN THE
the
The interassay variance was 6.5% and intraassay. variance 3.5%. All samples of one experiment were estimated in the same assay. FSH. Plasma FSH was determined by a validated homologous radioimmunoassay system, using an
as pig
age.
AND
METHODS
Procedures
landrace sows, mated on a single occasion, in these studies. Term is at 114 ± 1 days. experiments. Twenty-one sow, were aneswith azaperone (1.5 mg/k(’ i.m.) and (5 mg/kg iv.) (Janssen Pharma-
ceutica). The precise doses according to the reaction of Through a lateral abdominal uterus was exposed. Thereafter, cord was exteriorized through
of anesthetics varied the individual animals. incision, part of the part of the umbilical a small uterine incision.
The procedure up to the start of the sampling lasted 10-15 mm. A group of 3-6 fetuses per sow was studied; on two occasions a second group of 3-4 fetuses from the same sow was used. Blood samples (1.0-2.0 ml) were taken from the umbilical artery every 10 mm for 1 h. Synthetic LHRH (Hoechst) or physiological saline (1 ml) was injected into the umbilical vein 10 mm after the start of the experiment. The dose of LHRH used was 2 pg at 113 days, 1.5 pg at 100 days, 1 pg at 85 days, 0.5 pg at 70 days, and was calculated to supply 2 pg/kg estimated fetal body weight. Five males and 5 females were treated with LHRH per age group; a similar number of animals was treated with physiological saline. At 85 days, 3 male and 7 female fetuses were treated with physiological saline. Only one blood sample was taken from fetuses at 55 days of gestation, either before or I h after intramuscular injection of LHRH (0.25 Mg) or physiological saline, in a total of 15 fetuses. Chronically catheterized fetuses. Nine male and 1 female fetus (102 ± 1 days) from 8 sows were chronically catheterized and studied 3-4 days after surgery. Polyvinyl catheters were inserted into the fetal carotid artery and the jugular vein. Details of the surgery and animal care have been described previously (Macdonald and Colenbrander, 1980). All fetuses had normal arterial pH (>7.3) and P02 (‘19 mmHg) at the time of the experiment. Arterial blood samples were drawn every 10 mm for 1 h. LHRH (1.5 pg) or physiological saline (1 ml) was injected into the venous catheter 10 mm after starting the experiment.
Hormone LH.
Determinations
Plasma LH was determined by radioimmunoassay (Pomerantz et al., 1974). The anti-porcine LH serum (a gift from Dr. G. D. Niswender) was diluted 1:20,000 and 100 pI bound approximately 40% of the iodinated LH present in the absence of unlabeled LH. The standard LH preparation (LER-786-3, a gift from Dr. L. E. Reichert, Jr.) had a potency of 0.65 mg NIH-LH-S1/mg. The sensitivity of the assay was 0.2 ng LH/ml when 100 p1 of plasma was used. For purposes of statistical analysis, samples which were undetectable were arbitrarily assigned an average value of 0.1 ng/ml.
FETAL
immobilized and free sensitivity
PIG
557
second antibody for separation hormone (Van de Wiel et al., of the assay was 0.30 ng FSH/ml
of bound 1981). The when 200
p1 of plasma was used. The interassay variance was 10.0% and intraassay variance 8.1%. All samples of one experiment were estimated in the same assay. The standard FSH preparation (a gift from Dr. G. Hennen) had a potency of 1.56 mg LER-1419-3/mg. Statistical
Analysis
We have used simple analysis of variance such as one-way analysis of variance and of contrasts to detect statistical differences LH values at various ages between male fetuses, between treatments and between groups. For the statistical computations we use of a program from SPSS, a Statistical the Social Sciences (Nie et al., 1975).
techniques the method of FSH or and female surgery have made Package for
RESULTS
Plasma
Luteinizing
Hormone
At 55 days, plasma LH was at or below the detectable level (0.2 ng/ml) both before and after treatment with LHRH or saline. At 70 days, pretreatment concentrations averaged 0.5 ± 0.1 (mean ± SEM) ng/ml. Also, in the other age
groups
low 1.1
and average ng/ml (Fig.
pretreatment
concentrations
concentrations 1). As no
ferences
(P>0.05)
were
or
treatment,
the
after
averaged
per
trations plasma treated of
of 10
10 at
LHRH
0.7
days.
ng/ml
at
106
at
days,
70
days
and
significant
at
days.
days
plasma
ng/mI),
days,
in
all
older
4.3
in 7 out
fetuses response
observed
between
Plasma treatment
concenaveraged
1.8 at 100
difference
± 0.4
days,
± 0.7 in
ng/ml at 1.8 ± 0.3
ng/ml
at
the
response
between chronically those treated during
Follicle-Stimulating 55
1.0
85
maximal
was
ng/ml
be detected fetuses and
At
in
The
± 0.3
could terized
Plasma
and
1).
treatment
2.4
No
100
at
after treatment. 40 mm after LHRH
days,
ng/ml
days
LH>
(i
treated
mm
± 0.4
85
plasma LH conceninduced an increased in 2 out of the 10
at 70 days
(Fig.
20-40 trations
either before both sexes are Physiological saline for
time.
fetuses 100
(P0.05). LHRH LH concentration
treatment
remained significant
113
cathesurgery
Hormone FSH
concentrations
were
558
COLENBRANDER
ng LH/mI
ET
not
plasma
AL.
changed
ng/ml. 5-
4-
ment, from
113
/
3-
or sex (P>0.05).
-L days
2
/
100
days,
was
70
days
at
1.6
± 0.1
treatment
observed at pre-(LHRH)
70
days treat-
in females gradually at 85 days to 9.7
in
to
8.1
males
100,
2.3 ng/ml
106
2.0
±
rose ±
1.0
ng/ml
at
concentrations
and averaged 2.1 ± 0.4
85,
averaged of LHRH
and
± 0.3 and 113
re-
ng/ml, 4.3 3.2 ± 0.4
days,
respec-
tively.
-----
No
significant
observed.
In
0
LHRH
3
terized those
2).
/
/
after
16.0
V
saline
observed
was in
the
was
treatment
significant
effect
(Fig.
chronically
cathe-
treatment treatment
± 2.5
(P0.05)
males did not differ significantly from in 100-day-old male fetuses. All female (Fig. 3) older than 85 days responded to
LHRH
/
of no
Concentrations
mm 1
effect
males
injection
fetuses
2
3
100
treattreat-
DISCUSSION
2
1,
//1
In immature both within when
lOOdays
/6 / /
1
pigs
/ -1----,----!
85 days
of gestation. LH and
2
FSH
At mid do
treatment. In this rather undifferentiated.
1
70
days
(Liwska, response
1
0 I
0
1975). may
I
I
I
I
20
40
60
minutes
to LHRH reaching
a
1. Plasma pigs before estimated
LH concentrations (mean ± SEM) and after treatment with LHRH body weight, o---o) or saline
consistent
(.-.) in anesthetised (106 days) fetuses. Time the arrow.
and chronically of treatment
catheterized is indicated
by
end et
with
of gestation a!.,
a!.,
1974;
concentrations following
of LHRH
the pig pituitary Although growth
is hor-
be identified at 50 gonadotropic hormone visible until 70 days p.c. can
This indicates be due to
that the
gonadotropic increasing
age
the absence
lack of
of a
hormones in the response
finding that LH and FSH in the pituitary are higher at the than at 70 days p.c. (Melampy
1966).
The
increasing
LH
also of
be partly LHRH
due to receptors
such
is
the
LHRH could concentrations pituitary,
of
becomes more and more prominent, maximum at 113 days. This
concentrations FIG. fetal pg/kg
et
gestation not rise period
releasable pool of the pituitary. With
‘I
a release
1978b; Van de Wiel et a!., pig a gradually increasing can be seen at the end
mone producing cells days post coitum (p.c.), producing cells are not
0
induces
(Pomerantz
Colenbrander et a!., 1979). In the fetal response to LHRH
1_
0
LHRH
LH and FSH with a maxima! response 20 mm. This response can be observed LHRH is injected intravenously as well as
intramuscularly
/ 6--
in (2
days
and
while
lower ng/ml,
ng/ml
1’
1
at
mained ± 0.2,
/
and effect
concentrations 3.8 ± 1.0 ng/ml
113
/
significant difference After
ng/ml
/
by treatment
No
as
is observed
in
the
response
to
increased in the
neonatal
rat
RESPONSE
TO
LHRH
IN THE
(Chan
FETAL
PIG
et a!.,
1981),
increased development,
FSH/ml
ng
human
plasma
559
or
it may
be
related
to
an
endogenous LHRH secretion during as suggested for the sheep and
fetus
(Kaplan
et a!.,
1976;
Mueller
et a!.,
1981). 6-
113
days
In
the
sheep
fetus
the
effect
LHRH
4-
plasma gonadotropic hormone concentration maximal at mid gestation (Mueller et a!., In both human and sheep fetuses this
2-.
declines about
0-
end
towards term. This a negative feedback
by
estrogen
6_
106
days
and
of
term
Macdonald
2-
-
----H
.--.---
few
6-
al.,
at 1977;
a!.,
et
1979;
Silver
exerting
estradiol also rise a!.,
negative
response. Androgens a decline in LH concentrations
weeks
after
birth
a!.,
effect indeed do until a
(Colenbrander
et
a!.,
Ford et a!., 1978). This probably why a significant sex difference in the does not exist in pigs-at least with the of animals we used-as it does in
humans and sheep, where the increase LH concentrations following LHRH is higher in females than in males
4-
1978a; 1979),
a!., et
a
the
Mueller
LHRH
1978a; explains response number
0-
et
the pig, plasma Leydig cell activity
without
the cause
is probably brought due to increasing
(Colenbrander et
however, on not
In and
is 1981). effect
concentrations
(Takagi
et al., 1981). concentrations towards
4-
androgen
gestation
on
1977;
Mueller
et a!.,
in plasma treatment (Takagi et
1981).
2100
days
0-
The response is not markedly anesthesia, as no significant observed between anesthetised catheterized
6-
fetuses
concentrations. 85 days
4-
I
FSH to
levels
also 70 days
2
greater
above,
this
be
seem
sex
caused to
react
in the
on
the
in A
is
gestation
and
sheep
with males (Takagi 1981). As indicated
difference androgens,
LHRH males.
in
human
a!.,
or FSH
increased
with
at mid
female
by
not
Thereafter, basal significantly in with increased
response
in
observed
LH
was
treatment of response
lack
fetuses when compared a!., 1977; Mueller et to
0
to
the
significantly
0-
plasma
FSH
by LHRH before 85 days p.c. concentration of FSH increased females. The females responded plasma contrast
2-
in either
Plasma
influenced by differences were or chronically
pig
since
high
et
is unlikely
LH
androgen
does
level
not in the
male. 0
‘
20
60
40 minutes
FIG. 2. Plasma FSH concentrations (mean ± SEM) in fetal male pigs before and after treatment with LHRH (2 pg/kg estimated body weight, o---o) or saline (s-.-.) in anesthetised and chronically catheterized (106 days) fetuses. Time of treatment is indicated by the arrow.
In
the
adult
feedback on by granulosa a!., in
1977; the
mental Oxender
Jong, of
of
the
pig
a!.,
Inhibin
negative
do
(Mauleon 1979).
In
not et
the
et
production
is unlikely
oocytes
follicles et
part
1979).
female
stages
secondary
least
FSH is exerted by inhibin produced cells or Sertoli cells (Welschen
Dc
fetal
at
fetal
as
develop-
go
beyond
al., male
1978; pig,
560
COLENBRANDER
ET
AL.
Sertoli
FSH/mI
ng
cells
markedly
develop
at
the
end
of
gestation resulting in increased tubular length growth (Van Straaten and Wensing, 1977). Production of inhibin by the fetal Sertoli cells may provide an explanation for the lack of effect of LHRH on FSH in the males.
plasma
20-
ACKNOWLEDGMENTS
/
16-
I /
We would like to thank Mrs. U. Beermann, Mrs. A. Schulte-Derne, Mrs. C.M.J.E. Van Rossum-Kok and Mr. J. Erkens for their expert technical assistance. The authors wish to thank Dr. J.A.J. Faber for his statistical advice. This work was supported by a NATO Science Fellowship to B.C. granted by the Netherlands Organization for the Advancement of Pure Research (Z.W.O.).
\/
/
12-
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L
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