male rats do not show this self-priming effect. To examine some of the ... in male rats using serum collected from decapitated animals for hormone ... Castration.
BIOLOGY
OF
REPRODUCTION
29,
912-918
(1983)
Serum and Pituitary Luteinizing Hormone and Serum Androgens During Luteinizing Hormone Releasing Hormone Self-Priming in Immature and Pubertal Male Rats J.
S.
NAZ1AN
Department
of
College
Physiology
of Medicine
University
of
Tampa,
South
Florida
Florida
33612
ABSTRACT Pubertal
and
young
adult
male
rats
release
more
luteinizing
hormone
(LH)
in
response
to
luteinizing hormone releasing hormone (LHRH) if they are pretreated with LHRI-I than if they are pretreated with saline. Immature male rats do not show this self-priming effect. To examine some of the possible causes of this difference, immature or pubertal male rats were anesthetized with ketamine HCI and received 3 i.v. injections of 10 ng/100 BW LHRJ-I or saline at 30-mm intervals (Times 0, 30 and 60 mm) and they were decapitated at Times 0, 15, 30, 45, 60, 75 and 90mm. Serum and pituitary LH and serum testosterone and androstenedione were determined by radioimmunoassay. A self-priming effect was apparent in pubertal rats. LH levels in response to the third LHRH injection were significantly higher compared to the response to the first injection. No self-priming effect was evident in immature rats. No changes in pituitary LH that could account for this difference were apparent. Serum testosterone levels in response to the LHRH priming were relatively higher in immature rats than in pubertal animals at 30, 45 and 60 mm. Serum androstenedione levels were relatively higher in pubertal rats at 45 mm. These data indicate that LHRH self-priming effect can be demonstrated in male rats using serum collected from decapitated animals for hormone analysis. They also suggest that a different relative response of serum androgens may be one cause of the appearance of LHRH self-priming during the sexual maturation of the male rat. INTRODUCTION One
the
of
course
the changes that of sexual maturation
is the development luteinizing hormone
takes
of a self-priming releasing hormone
(Nazian and Mahesh, adult male rats release
1980b). more
development of this effect known. Castration eliminates effect (Nazian and Mahesh,
is not currently the self-priming 1979b). Pretreat-
effect of (LHRH)
ment
can
or young hormone
and
place during in the male rat
Pubertal luteinizing
LHRH
(Nazian develops show and
challenge
regardless
and Mahesh, between 34 no
46
statistically The
evidence days of factor
of any age, when
significant (Nazian, 1981). or factors responsible
testicular effect
1980a).
These
June 20, 1983. March 21, 1983. requests: Dr. Stanley
the
effect
(Nansel
Physiology, College of Medicine - Box South Florida, Tampa, FL 33612.
Nazian,
Dept.
8, University
to
suggests the steroid response
in
be
part,
immature
responsible rat
to
al.,
of
greater
of
than more
912
inhibition is that
effect
on
1979).
the
failure
a self-priming
latter
could result. An priming injections
LH
synthesis for
of
the
effect.
If
during the priming rats, then a greater
the
in immature males. LH being available
This
possibility that the during priming may, for
show
preeffect that
have a rapid direct to modify the self-
et
observation dynamic
degree of possibility
J.
suggest
steroids in the rise in serum appears
androgens can the pituitary
on
the (Nazian
observations
more testosterone is released process in immature male Accepted Received ‘Reprint
prevent effect
of testicular the pubertal concentrations
priming
effect, is first for
Mahesh,
self-priming
cede the expression of the self-priming (Nazian, 1981). It has also been suggested
This effect age, when rats
self-priming the effect
of the
testosterone
of pretreatment
1979b). days of
androstenedione
the involvement process. Indeed,
(LH) in response to a LHRH challenge if they are pretreated with LHRH than if they are pretreated with saline. Immature male rats release essentially that same amount of LH after
with
development
in older
alternate have
a
animals
This would result in release. The experi-
HORMONE
ments these
reported
here
were
questions.
pubertal stages
rats were priming.
single
large
weaning from were maintained
group
the
of
of
immature
and
at
rats
was
WI).
at
They of tem-
(14L:1OD) with food and They were housed 4-6 per cages. Developmental stages
were as defined previously (Nazism and Mahesh, 1979a,b). Immature rats were used at 29, 30 or 31 days of age; pubertal rats were used at 49, 50 or 51 days of age. In this colony, based on such criteria as secondary sexual organ growth, negative feedback sensitivity and serum testosterone concentrations, puberty begins about Day 40 and is essentially complete by Days 65-70. All animals were anesthetized with ketamine HCI (10-20 mgJlOO g BW) i.p. supplemented with additional i.m. injections as needed (Nazism, 1981). Beginning at 0800-0900 h rats received 3 i.v. (jugular) injections of 10 ng/100 g BW LHRH or saline at 30-min intervals. Synthetic LHRH (lot No. 21-103-DH) was obtained from the National Pituitary Agency and the NIAMDDK. Groups of 6 rats were decapitated at Time 0 (prior to any injection) 15, 30, 45, 60, 75 and 90 min. LHRH or saline injections were given at Time 0, 30 and 60 min. Serum was frozen until subsequent determination of LH, testosterone and androstenedione by radioirnmunoassay. Pituitaries were carefully dissected, the posterior lobes removed and the anterior lobes weighed and individually stored frozen in 1 ml of 0.9% saline. Subsequently they were thawed and homogenized in 2 ml of phosphate-buffered saline (pH=7.5) (PBS) with a Teflon glass homogenizer. Glassware and the Teflon pestle were washed with the storage saline and an additional 1 ml of PBS. All washings and the homogenate were combined and centrifuged at 3000 rpm for 15 min. All procedures were carried out at 0-4#{176}C. The supernatant was refrozen until assayed for LH by radioimmunoassay.
TABLE
1. Hormone
levels
at Time
0 (Mean
±
LH
10.9 LH
content
LH
concentration
913 and
Statistical
Analyses
pituitary LH were determined by using an antibody (#15) obtained Niswender (Niswender et al., 1968) rat LH (Nazian, 1981). LH values by and are expressed in terms of the RP-1. Serum concentrations of androstenedione were also determined by radioimmunoassay using previously published techniques (Nazian, 1981) Data were analyzed and significance determined using Student’s t test for comparisons between two groups and Duncan’s new multiple range test for multigroup comparisons.
RESULTS
Serum prior
and
to the
age
pituitary
start
groups
are
LH
were compared
rats
testosterone higher in
tive
Time
Serum
Table
1.
levels
just
in the
two
Serum
significantly and pituitary
levels pubertal
were rats.
LH
different. concen-
significantly to immature
significant difference dione concentrations Due to these differing analyses pituitary first calculated
priming
in
were not content
of
Serum 10-fold
hormone
LHRH
given
concentrations Both pituitary tration pubertal
of
higher
in males.
approximately There was
no
in the serum androstenebetween the two ages. baselines, in subsequent
LH and serum as the percentage
steroids of the
were respec-
0 value.
LH
(Fig.
1)
Treatment ng/100 g
BW
(P0.05
49.9
