equivalent to females on Days. 17.5-19.5. (Charpenet et al., 1982;. Salisbury ...... rats. Neuroendocrinology. 25:247-56. Greenwood. FC,. Hunter. WM,. Glover. iS,.
BIOLOGY
OF
REPRODUCTION
Plasma
40, 111-117 (1989) Luteinizing Hormone
Levels in Normal
Male and Female RONALD
SALISBURY,3’4
Rat Fetuses
JONATHAN
REED,5
Department The
and Their
INGEBORG
of Obstetrics Milton
The
and
S. Hershey
State
JUDITH
WEISZ4
Center
University 17033
of Psychology5
Villan Villan
and
Gynecology4
Pennsylvania and
Department
Stressed
Mothers’
L. WARD,2’5
Medical
Pennsylvania
Hershey,
and Prenatally
ova
ova,
University
Pennsylvania
19085
ABSTRACT
from
Concentrations of luteinizing control mothers and from
stress used the and and
to be
is known
associated
fetal testes. The overall ontogenetic female rats, and was unaffected then rose progressively through
the already low levels of LH stressed fetuses of both sexes both the control and stressed tions
hormone mothers
of females
exceeding
those
with
group
an
abnormal
pattern by stress. birth, i.e.
between with
(LI-I) were measured in plasma of fetal and neonatal rats obtained exposed to stress from Days 14 to 21 of gestation. The regimen of
16 and levels below
became
of males
from
evident Day
21
developing
nervous
system
ontogeny. development
In rats, increases
of
tial for female sexual behavior, ability to display male patterns 1984). We have shown that male stressed during titers of plasma steroid
pregnancy testosterone
dehydrogenase
during stress the and (see fetuses
critical
stages
activity
gestation,
but
lack
steroidogenic
enzyme
levels
found
in
(Orth 1984;
and Weisz, Weisz and
the
normal
findings
zation
and
impairs their review Ward, from mothers
patterns changes
of prenatally in the ontogenetic
activity
of
Day
suggest
from
the
fetal
both
blood
on
Ward 1980;
that
in
and
Days
18
testes
which,
behavioral result from steroidogenic
in turn,
testosterone. of Leydig
19
1980, 1983).
masculini-
sexual
stressed males pattern of
of plasma secretion
and
Weisz, et al.,
incomplete of
testicular testosterone
and Orth
the
defeminization
abnormal pattern Testosterone
17
surge
males
1980; Ward,
These
on
secretion
activity
experienced males’ poten-
have higher than normal and testicular t -35-ol
(313-HSD)
testosterone
levels in plasma was similar in male was low from Days 16 to 20 of gestation, stressing the mother significantly decreased
to 23 post-conception.
The extent to which the sexual behavioral potential of male rodents is masculinized and defeminized depends on the amount of testosterone available to of perinatal during fetal
of
LH
20, as indicated by a larger percentage of samples from the limit of sensitivity of the assay. Sex differences in only after Day 20 of gestation, with plasma concentra-
INTRODUCTION
the
pattern
immunoreactive In all groups, LH Day 23. However,
Days LH
ontogenetic
of
cells
lead in
to an rats
is
thought to come under the control of pituitary luteinizing hormone (LH) during the last week of gestation. Therefore, the present study was undertaken to assess whether the changes in plasma testosterone levels and testicular steroidogenic enzyme activity that characterize stressed rat fetuses are associated with altered levels of plasma LH. In
Accepted August 11, 1988. Received July 31, 1987. ‘This study was supported by Grant HD00954 (to J. W.), and by Research Scientist Award 2-K05-MH00049 from the NIMH and Grant HD04688 from the NICHHD (to I. L. W). 2 requests. address: Department of Biology, University of Akron, Akron, OH 44325.
111
addition, females
we since
measured there are
regarding fetuses.
the Males
ontogenetic have been
LH levels in control males and discrepancies in the literature pattern reported
of plasma LH in rat to have levels of LH
SALISBURY
112 higher
than
females
on
Days
16-20
of
gestation
(Chowdhury and Steinberger, 1976), equivalent to females on Days 17.5-19.5 (Charpenet et al., 1982; Salisbury et al., 1982), or lower than females on Days 19-21 Picon,
(Slob 1982).
were
taken
and
control
nancy
et al., 1980) or 20.5-2 In the present study, from
male
and
mothers
and
from
post-conception
female
killed their
on
1.5 (Habert and plasma samples fetuses
Days
newborn
of stressed
16-22
pups
Day
23
(pc). MATERIALS
AND
same sex, of 0.3 ml
age, and treatment was pooled to a volume per sample. On Days 22 and 23, the blood
from a single pup provided 16 and 17, in most cases, mates
of
sample. not
of preg-
on
ET AL.
the
same
Plasma
pooled.
The litters of 121 nulliparous female rats (Harlan Sprague-Dawley, Madison, WI), mated between 70 and 90 days of age, were used. All animals were housed in a temperature controlled vivarium (23#{176}C), on a reversed day-night cycle (lights h). Purina rat chow (Ralston-Purina, MO)
and
water
were
available
off, St.
ad libitum.
Procedure Estrous males
females
in the
were
middle
mated
of the
by
dark
h) until noon of
two ejaculations were mating was considered
Half
the
of
control The
animals
group animals
X 5 X 8 cm delphia, PA), (2150 lm/m2) (Ward, 1972). ning on Day killed at an of
gestation.
1700
placed
with
(1100-1300
observed. The afterDay 0 of gestation.
assigned
and half to the were stressed
randomly
to
stress group. by being placed
into
the 13
Plexiglas holders (A. H. Thomas, Philailluminated by two 150-W floodlights for 45 mm at 0900, 1300, and 1700 h This treatment was given daily begin14 of gestation and, earlier stage, continued Control
they were killed. Mothers from Days 16 through mm
were
being
period
animals
were
each treatment 22 of gestation,
h. The stressed after the second
mothers stress
unless a group was through Day 21 not
handled
group were between
were session
until
killed on 1300 and
killed at least 45 of the day. The
pregnant animals were decapitated, after being stunned by a blow to the head, and the trunk blood was collected in a test tube that was centrifuged for 5 mm. The serum was flash-frozen in a bath of methanol and dry ice. Plasma was collected from fetuses and neonates as described previously (Ward and Weisz, 1984). alizing
The the
sex of each gonads. The
fetus was determined plasma from animals
by visuof the
needed
fetuses
samples
of
were of assay. obtained
neonatal
pups
animals.
Only
by
an
Dodge Laboratories, with the litter. The from
the nest
and
also
born
of Ft. were
plasma
a
litters and from
h the
Chioropent
samples
of
23 pc were of birth. mother
Dodge, removed
was stored
groups
on Day 12
injection
killed;
complete
different
within neonates,
Inc., pups
to
On Days all litter-
flash-frozen
at -70#{176} C until the time Plasma samples were
anesthetized
Louis,
was
used. They were killed minimize stress to the
METHODS
Animals
maintained 0800-2000
sex
from
The
a 0.3-ml sample. the plasma from
To was (Fort
IA) and kept individually were
collected
as for the fetuses. Maternal samples were not collected. The titers of LH were measured using radioimmunoassay with kits supplied by the Rat Pituitary Distribution Program, NIAMDD. The microassay method of Coquelin and Bronson (1980) was used with slight modification. For assay of fetal and neonatal samples, LH (NIAMDK LH 1-5) was iodinated to approximately 100 jiCi4zg using chioramine T (Greenwood et al., 1963). The 75
mm
following
borosilicate
overnight
incubations
of LH (NIAMDK (BSA)-phosphate-buffered .il of plasma
at room RP-1)
to 10 X
reagents were added
test tubes
in two
consecutive
temperature:
(1)
50 j.zl
in 1% bovine serum albumin saline (PBS) buffer, or 20
in 30 zl of buffer,
plus
50ti
of NIADDK
anti-rat LH S-4 at 1:15,000 dilution in 0.1 M ethylenediaminetetraacetate (EDTA)-PB S without normal rabbit serum; and (2) 50 p1 [‘25I]-LH (NIAMDK LH 1-5) in 1% BSA-PBS at 20,000 dpm. On the third day, 100 p1 of Immunobead goat anti-rabbit immunoglobulin (BioRad Laboratories, Richmond, CA) were added and the tubes were incubated for 6 h at room temperature. After the addition of 2 ml 0.1 M PBS was
and centrifugation, the radioactivity in each tube counted in a gamma counter (Beckman Instru-
ments). The LH concentrations were expressed as nanogram equivalents of NIAMDK LH RP-1. The assay procedure for the maternal samples was identical to that used for the fetal and neonatal samples with the exception the assays of the maternal was used as the iodination LH-S-7 rLH-RP-2
(1:7000) as
as the the
of the reagents samples, NIADDK standard, NIADDK first
reference
antibody, standard.
and
used. In rLH-I-6 anti-rat NIADDK
The
values
PLASMA obtained for the maternal samples ally corrected so that they could terms of NIADDK rLH-RP-1. The cient
of
variation
and
fetal
was
measured
for
samples
the
was
and
in all of the
in a single
assay,
as were
sensitivity
of the
fetal
was
of
3.3%,
maternal LH
neonatal
from 0.16
the
respectively.
and
those
assay
were mathematicbe expressed in intraassay coeffi-
assays
2.9
LH IN FETAL
the
The
ng LH-RP-1/tube
analysis U-tests. lower Days
(8
ng/ml).
this
group are 16-day-old were
for
for and each
shown in Table 1. Of the 67 samples from fetuses, 56% were assayed in duplicate, as
all but
maining values
samples assayed combinations samples to
27
of the
505
samples
groups. Only measurements duplicate samples differed
from
the re-
for which by less than
the 20%
either
time
across
rose
when males
There
in
warranted, and females
for
LH
by Mann-Whitney had significantly
of the
decreased and
collapsed
changes
between stress
this
fetuses
on
during collapsed
across
Days
groups
days.
16 and
(p