Plasma Luteinizing Hormone Levels in Normal and Prenatally ...

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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