BIOLOGY
OF
REPRODUCTION
Plasma
17,
313-320
(1977)
Levels of LH and FSH in Female Rats with Precocious Puberty Induced by Hypothalamic Lesions’ J.
P. ADVIS2
Instituto
and
V.
de Fisiologia
D.
RAMIREZ3
Facultad
Universidad
Austral Valdivia,
de Medicina, de Chile,
Chile
ABSTRACT precocious puberty induced associated with alterations in plasma gonadotropin levels were made by radiofrequency in the anterior hypothalamus levels of gonadotropins were determined by radioimmunoassay To
determine
whether
following the lesion. Lesioned animals
Nonoperated showed
and precocious
sham-operated puberty
as
by anterior hypothalamic during the prepubertal of 15 day old female (RIA) at different
littermates evidenced
lesions was period, lesions rats and plasma *ime intervals
were used as control. by early vaginal opening
and
first
ovulation. They also became pregnant at an earlier age than controls and delivered their pups normally. Lesioned animals showed a more pronounced rate of increase in ovarian weight during the first 15 days following the lesion than that of control rats. Nevertheless, plasma FSH levels were not different in both groups. Plasma LH levels during the morning of the days preceding vaginal opening were somewhat higher in control rats than in lesioned animals. However, during the afternoons LII levels tended to be more elevated in lesioned rats than in controls, the difference being significant at Day 23. Estradiol injected subcutaneously inhibited compensatory ovarian hypertrophy in lesioned rats demonstrating the integrity of the estrogen negative feedback mechanism. The results were interpreted as not supporting the view that hypothalamic lesions induce precocious puberty by eliminating negative feedback to estrogen. The possibility arises that precocious puberty induced by hypothalamic lesions is determined by other mechanisms and an alternative explanation is suggested.
INTRODUCTION In
1956,
Bosch
Donovan
reported an female
final
event rat.
center
der
in
the
Results
were
of
destruction
Werff
the
age
associated
consequence
thalamic
van
lesions
advanced
opening, the
and
that
hypothalamus
and
with
ten
puberty
of
inhibitory
for
as some
the
hypo-
pituitary
FSH
Since lesions
then in
investigated Werff 1967;
precocious
the
anterior by
ten
Bosch,
Meijs-Roelof
puberty
induced
hypothalamus
many
has
(Donovan
and
1959;Critchlow and
Moll,
and 1972;
by been
van
der
Bar-Sela,
are
present
(Eisenfeld
and
Axelrod,
and
McEwen, precocious
lesions
mic
feedback
2Present address: Department ncr Hall, Michigan State University, 48824. 3Present address: Department Biophysics, University of Illinois,
of
Physiology, East Lansing,
in female
rats
duced
by
anterior
lesions
resulted actions
pituitary
secretion
Preliminary
1973a,
313
in b).
of
work
with
abstract
is
estradiol
to
report
levels
precocious
estrogen
some
the imma-
puberty
in-
If the
of hypothalamic to
the
then,
somehow
form
in
lesions.
sensitive
of
been
hypothalato
hypothalamic
reports
appeared
of Physiology and Urbana, IL 61801.
has
1974).
this
of
Vile, 1975;
following
destruction
in destruction
feedback
it
puberty
gonadotropin
centers
and 1970,
sensitive
of
plasma
hypothalamus
Kato,
Donovan,
purpose
inhibitory GiltMI
to
in
estradiol
Kato
1973),
receptors 1971;
The
ture
is due
that
the
1972;
that
con-
secretion
1967;
Plapinger
gonadointo
hypothalamic
and
in
accepted these
anterior
1954)
al.,
et
for
FSH
(Flerko,
Ginsburg
a common
now
Taking
pituitary
receptors
changes Accepted April 20, 1977. Received August 18, 1976. ‘This work was supported in part by a Population Council Grant to V. D. Ramirez, and by Vicerectoria de Investigaciones, Univ. Austral de Chile.
the
of
rats
(Ramirez,
Sherwood
effects
on
is
center
destroyed.
parabiotic
1967;
secretion.
is
the
it
inhibitory
secretion
lesions
in
and
some
sideration
vaginal
interpreted
1974)
that
tropic
anterior of
Timiras,
belief
must of (Advis
negative
LH
and
be
these and
FSH
altered.
results Ramirez,
has
ADVIS
314
MATERIALS Female
albino
AND
rats
belonging
AND
separately until delivery. body weights were recorded.
METHODS to
a
RAMIREZ
Wistar-Holtz-
mann mixture colony inbred in our laboratory for at least 12 years were used in these experiments. Rats were housed under controlled conditions of temperature (24#{176}-26#{176}C)and light (14 h on, from 0500 to 1900 h). Animals were given ad libitum access to food (Cooperative Agricola de Purranque) and water. At 15 days of age rats were weighed and individually identified. Some animals were lesioned bilaterally in the anterior hypothalamus and returned to their mothers. Littermates were used as intact nonoperated control, or sham-operated control (animals in which the electrode was just lowered to the cortex and then drawn out). All animals were weaned at 20 days of age.
Experiment
Lesions
Animals
positioned in a rat stereotaxic Kopf Instruments) and lesions were made by a 2M Hertz square wave current (.020 A/20 seconds) generated by a radiofrequency (r-f) lesion maker (Grass, model LM3) through a unipolar (26 gauge) stainless steel electrode (anode) completely isolated with epoxylite resin except for 1 mm at the tip. Voltage was monitored throughout the process with a multitester (Simpson, model 260, 5 kIl/volt)
apparatus
were
(David
connected in parallel with the lesion cathode was connected to the stereotaxic The lesion was localized 1.1 mm
marker.
The
apparatus. anterior to bregma, 0.6 mm lateral from midline and 7.8 mm below the external surface of the cerebral cortex. Preliminary average approximation at this site, based on the DeGroot Rat Stereotaxic Coordinates Manual (1959) and histological methods, helped to select the coordinates. At the end of each experiment, brains were removed and fixed in 10 percent formalin for at least 48 h. Frozen frontal sections (80 p) were made in order to determine the site of the lesion.
Experiment
1
Control animals and their lesioned littermates were sacrificed the day of their first estrus (between 1000 h and 1200 h). Anterior pituitary and ovarian wet weights and ovulation (detected by direct microscopic observation of ova in the oviduct) were determined.
Experiment
2
Control and lesioned littermates were caged with males of proven fertility (‘ 4 months old) on the day on which their vaginas were about to open (vaginal edema or vaginal membrane partially open). Each female rat (control or experimental) remained housed with the male up to its first metestrus. Males were caged first with a lesioned rat and after 3 days of rest, with control littermates. Sexual receptivity was controlled by the detection of spermatozoa in the daily vaginal smear. From that day each rat was kept
of
pups
and
their
3
Blood (0.4 ml) was obtained under light ether anesthesia using heparinized syringes, from the external jugular vein between 0800 h and 1400 h from control and lesioned rats of 16, 20, 30, 35, 40 and 45 days of age. Blood samples were processed as detailed elsewhere (Ojeda and Ramirez, 1972) and plasma gonadotropins were determined by RIA. In one experiment each control or lesioned animal of 1 7, 23 and 30 days of age was bled at 0800, 1600 and 2400 h for determination of plasma LH. Blood samples were obtained from the left jugular vein (0800 h), right jugular vein (1600 h) and from jugular vein or sometimes
Hypothalamic
Number
by
last bleeding exsanguination. dissected
puncture the animals
Pituitary out
(precision examination
animals
cardiac (2400 h) and
glands
weighed
0.005 g). of ovaries
±
in
(2400 were
and a
h). After sacrificed
the by
ovaries torsion
In some cases, from control
were balance
histological and lesioned
was performed.
Experiment
4
Immediately after lesions were made, control and lesioned rats had their left gonad removed and their wet weight recorded. On the day of hemicastration, all animals received one sc injection of 0.05 pg or 0.5 pg of estradiol valerate (E2-V; Primofol Depot, Schering Berlin). They were sacrificed 24 h later and the remaining ovary was dissected out and its weight determined. Percent of 24 h compensatory ovarian hypertrophy (CO H) was defined as: =
l(right
ovary)
-
(left
ovary)l
/(left
ovary)
X 100 Radioimmunoassay LII and FSH were assayed in plasma samples of 0.15-0.20 ml, using reagents supplied by NIAMDD’l. When samples were less than 0.20 ml they were made up to this volume with phosphate buffered saline solution (pH 7.5) containing 1 percent bovine serum albumin (Sigma). To avoid interassay variation, control and experimental samples were run in the same assay. The minimum quantity of LH detected in our system was 14 ng. The highest values were 700 ng. Minimum quantity of FSH detected was 20 ng and the highest, 800 ng. Linear portion of the standard curve was between 20-80 percent precipitable counts for LII and 25-86 percent for FSH. Results were expressed in terms of the RP-1 standards supplied with the kits. Statistical analysis of results was made using the Student’s t test. In some cases the x2 test (2 X 2 using the Yate’s correction) was utilized. All statistical analysis were performed using a Hewlett-Packard desk computer, model 9100 A.
RESULTS 4The NIAMDD supplied by National bolic and Digestive Distribution Program.
standard Institute Diseases
hormone of Arthritis Rat Pituitary
was kindly and MetaHormone
Figure
depicting lesions
were
1
shows
the
sites included
a
diagram
where in
of
a
lesions
were
a zone
posterior
rat
brain
All
made. to
the
BRAIN
4’
..
LESIONS,
SEXUAL
MATURATION
AND
GONADOTROPINS
315
..,
E
//
#{149} eS:o.tr
I
0
RATS
CONTROL
a
RATS
a POA
-.
2
a.
a 0
30
E 20
FIG. 1. Saggital section of the rat brain showing the area where radiofrequency lesions were made. HP = Pituitary gland, AHA = Anterior hypothalamic area, POA = Preoptic area, SC = Suprachiasmatic nucleus, VMH = Ventromedial nucleus, PVH = Paraventricular nucleus, ARH = Arcuate nucleus, CO Optic chiasma. Dashed zone indicates the area in which all the lesions were found. Usually each rat had at least 50 percent of this zone destroyed.
POA,
delimited
of the der of portion
Table
with
1,
puberty
Lesioned
weights
a lower
animals
was
same
in
was
advanced
Also
body
Nevertheless, was
same.
between
controls
the were
in
controls
in
Figure
the 2
weights to
these
from
was
45
days. a
first
delivery
lesioned was
and
Pituitary lesioned
30
35
IN
40
45
weights Numbers
in in
DAYS
gland and ovarian rats at various ages.
Since
intact
differ-
and
and
lesioned
Twenty-four
by
used
and
animals h after
from
the in
lesion
pituitary
from
lesioned
higher
than
that
of
30. samples
were
plasma
were
rats
during
experimental (Fig.
3).
Plasma
and
in
general
higher
in control
rats
taken
levels
studied
out
fol-
weight
variable
and
ones
weight lesioned
quite
(Fig.
age
and
period
than
in
the
similar the
LH
in entire
levels
were
tended
LH levels
to
be
in lesioned
3). animals 1600,
30
and
days,
lesioned gland
Day
of
ovarian pituitary rats were
control
control
ovarian
FSH
(0800,
as
in
pitui-
days
increased 25 days, lesioned the
was in
pituitary
significantly
blood
When
puberty
were
still
by At
of
trends
controls
difference at 20
thereon
However, was
morning,
control
pituitary
decrease
rats
those
similar
intact
rats. from
From
lowed
rats
lesioned
of
to This present
still
than
control
(see
intact
was
When in
experiments.
ovarian
glands
controls
litterweight
rats
tary
group.
sham
from
compared (P
OF
REPRODUCTION
Plasma
17,
313-320
(1977)
Levels of LH and FSH in Female Rats with Precocious Puberty Induced by Hypothalamic Lesions’ J.
P. ADVIS2
Instituto
and
V.
de Fisiologia
D.
RAMIREZ3
Facultad
Universidad
Austral Valdivia,
de Medicina, de Chile,
Chile
ABSTRACT precocious puberty induced associated with alterations in plasma gonadotropin levels were made by radiofrequency in the anterior hypothalamus levels of gonadotropins were determined by radioimmunoassay To
determine
whether
following the lesion. Lesioned animals
Nonoperated showed
and precocious
sham-operated puberty
as
by anterior hypothalamic during the prepubertal of 15 day old female (RIA) at different
littermates evidenced
lesions was period, lesions rats and plasma *ime intervals
were used as control. by early vaginal opening
and
first
ovulation. They also became pregnant at an earlier age than controls and delivered their pups normally. Lesioned animals showed a more pronounced rate of increase in ovarian weight during the first 15 days following the lesion than that of control rats. Nevertheless, plasma FSH levels were not different in both groups. Plasma LH levels during the morning of the days preceding vaginal opening were somewhat higher in control rats than in lesioned animals. However, during the afternoons LII levels tended to be more elevated in lesioned rats than in controls, the difference being significant at Day 23. Estradiol injected subcutaneously inhibited compensatory ovarian hypertrophy in lesioned rats demonstrating the integrity of the estrogen negative feedback mechanism. The results were interpreted as not supporting the view that hypothalamic lesions induce precocious puberty by eliminating negative feedback to estrogen. The possibility arises that precocious puberty induced by hypothalamic lesions is determined by other mechanisms and an alternative explanation is suggested.
INTRODUCTION In
1956,
Bosch
Donovan
reported an female
final
event rat.
center
der
in
the
Results
were
of
destruction
Werff
the
age
associated
consequence
thalamic
van
lesions
advanced
opening, the
and
that
hypothalamus
and
with
ten
puberty
of
inhibitory
for
as some
the
hypo-
pituitary
FSH
Since lesions
then in
investigated Werff 1967;
precocious
the
anterior by
ten
Bosch,
Meijs-Roelof
puberty
induced
hypothalamus
many
has
(Donovan
and
1959;Critchlow and
Moll,
and 1972;
by been
van
der
Bar-Sela,
are
present
(Eisenfeld
and
Axelrod,
and
McEwen, precocious
lesions
mic
feedback
2Present address: Department ncr Hall, Michigan State University, 48824. 3Present address: Department Biophysics, University of Illinois,
of
Physiology, East Lansing,
in female
rats
duced
by
anterior
lesions
resulted actions
pituitary
secretion
Preliminary
1973a,
313
in b).
of
work
with
abstract
is
estradiol
to
report
levels
precocious
estrogen
some
the imma-
puberty
in-
If the
of hypothalamic to
the
then,
somehow
form
in
lesions.
sensitive
of
been
hypothalato
hypothalamic
reports
appeared
of Physiology and Urbana, IL 61801.
has
1974).
this
of
Vile, 1975;
following
destruction
in destruction
feedback
it
puberty
gonadotropin
centers
and 1970,
sensitive
of
plasma
hypothalamus
Kato,
Donovan,
purpose
inhibitory GiltMI
to
in
estradiol
Kato
1973),
receptors 1971;
The
ture
is due
that
the
1972;
that
con-
secretion
1967;
Plapinger
gonadointo
hypothalamic
and
in
accepted these
anterior
1954)
al.,
et
for
FSH
(Flerko,
Ginsburg
a common
now
Taking
pituitary
receptors
changes Accepted April 20, 1977. Received August 18, 1976. ‘This work was supported in part by a Population Council Grant to V. D. Ramirez, and by Vicerectoria de Investigaciones, Univ. Austral de Chile.
the
of
rats
(Ramirez,
Sherwood
effects
on
is
center
destroyed.
parabiotic
1967;
secretion.
is
the
it
inhibitory
secretion
lesions
in
and
some
sideration
vaginal
interpreted
1974)
that
tropic
anterior of
Timiras,
belief
must of (Advis
negative
LH
and
be
these and
FSH
altered.
results Ramirez,
has
ADVIS
314
MATERIALS Female
albino
AND
rats
belonging
AND
separately until delivery. body weights were recorded.
METHODS to
a
RAMIREZ
Wistar-Holtz-
mann mixture colony inbred in our laboratory for at least 12 years were used in these experiments. Rats were housed under controlled conditions of temperature (24#{176}-26#{176}C)and light (14 h on, from 0500 to 1900 h). Animals were given ad libitum access to food (Cooperative Agricola de Purranque) and water. At 15 days of age rats were weighed and individually identified. Some animals were lesioned bilaterally in the anterior hypothalamus and returned to their mothers. Littermates were used as intact nonoperated control, or sham-operated control (animals in which the electrode was just lowered to the cortex and then drawn out). All animals were weaned at 20 days of age.
Experiment
Lesions
Animals
positioned in a rat stereotaxic Kopf Instruments) and lesions were made by a 2M Hertz square wave current (.020 A/20 seconds) generated by a radiofrequency (r-f) lesion maker (Grass, model LM3) through a unipolar (26 gauge) stainless steel electrode (anode) completely isolated with epoxylite resin except for 1 mm at the tip. Voltage was monitored throughout the process with a multitester (Simpson, model 260, 5 kIl/volt)
apparatus
were
(David
connected in parallel with the lesion cathode was connected to the stereotaxic The lesion was localized 1.1 mm
marker.
The
apparatus. anterior to bregma, 0.6 mm lateral from midline and 7.8 mm below the external surface of the cerebral cortex. Preliminary average approximation at this site, based on the DeGroot Rat Stereotaxic Coordinates Manual (1959) and histological methods, helped to select the coordinates. At the end of each experiment, brains were removed and fixed in 10 percent formalin for at least 48 h. Frozen frontal sections (80 p) were made in order to determine the site of the lesion.
Experiment
1
Control animals and their lesioned littermates were sacrificed the day of their first estrus (between 1000 h and 1200 h). Anterior pituitary and ovarian wet weights and ovulation (detected by direct microscopic observation of ova in the oviduct) were determined.
Experiment
2
Control and lesioned littermates were caged with males of proven fertility (‘ 4 months old) on the day on which their vaginas were about to open (vaginal edema or vaginal membrane partially open). Each female rat (control or experimental) remained housed with the male up to its first metestrus. Males were caged first with a lesioned rat and after 3 days of rest, with control littermates. Sexual receptivity was controlled by the detection of spermatozoa in the daily vaginal smear. From that day each rat was kept
of
pups
and
their
3
Blood (0.4 ml) was obtained under light ether anesthesia using heparinized syringes, from the external jugular vein between 0800 h and 1400 h from control and lesioned rats of 16, 20, 30, 35, 40 and 45 days of age. Blood samples were processed as detailed elsewhere (Ojeda and Ramirez, 1972) and plasma gonadotropins were determined by RIA. In one experiment each control or lesioned animal of 1 7, 23 and 30 days of age was bled at 0800, 1600 and 2400 h for determination of plasma LH. Blood samples were obtained from the left jugular vein (0800 h), right jugular vein (1600 h) and from jugular vein or sometimes
Hypothalamic
Number
by
last bleeding exsanguination. dissected
puncture the animals
Pituitary out
(precision examination
animals
cardiac (2400 h) and
glands
weighed
0.005 g). of ovaries
±
in
(2400 were
and a
h). After sacrificed
the by
ovaries torsion
In some cases, from control
were balance
histological and lesioned
was performed.
Experiment
4
Immediately after lesions were made, control and lesioned rats had their left gonad removed and their wet weight recorded. On the day of hemicastration, all animals received one sc injection of 0.05 pg or 0.5 pg of estradiol valerate (E2-V; Primofol Depot, Schering Berlin). They were sacrificed 24 h later and the remaining ovary was dissected out and its weight determined. Percent of 24 h compensatory ovarian hypertrophy (CO H) was defined as: =
l(right
ovary)
-
(left
ovary)l
/(left
ovary)
X 100 Radioimmunoassay LII and FSH were assayed in plasma samples of 0.15-0.20 ml, using reagents supplied by NIAMDD’l. When samples were less than 0.20 ml they were made up to this volume with phosphate buffered saline solution (pH 7.5) containing 1 percent bovine serum albumin (Sigma). To avoid interassay variation, control and experimental samples were run in the same assay. The minimum quantity of LH detected in our system was 14 ng. The highest values were 700 ng. Minimum quantity of FSH detected was 20 ng and the highest, 800 ng. Linear portion of the standard curve was between 20-80 percent precipitable counts for LII and 25-86 percent for FSH. Results were expressed in terms of the RP-1 standards supplied with the kits. Statistical analysis of results was made using the Student’s t test. In some cases the x2 test (2 X 2 using the Yate’s correction) was utilized. All statistical analysis were performed using a Hewlett-Packard desk computer, model 9100 A.
RESULTS 4The NIAMDD supplied by National bolic and Digestive Distribution Program.
standard Institute Diseases
hormone of Arthritis Rat Pituitary
was kindly and MetaHormone
Figure
depicting lesions
were
1
shows
the
sites included
a
diagram
where in
of
a
lesions
were
a zone
posterior
rat
brain
All
made. to
the
BRAIN
4’
..
LESIONS,
SEXUAL
MATURATION
AND
GONADOTROPINS
315
..,
E
//
#{149} eS:o.tr
I
0
RATS
CONTROL
a
RATS
a POA
-.
2
a.
a 0
30
E 20
FIG. 1. Saggital section of the rat brain showing the area where radiofrequency lesions were made. HP = Pituitary gland, AHA = Anterior hypothalamic area, POA = Preoptic area, SC = Suprachiasmatic nucleus, VMH = Ventromedial nucleus, PVH = Paraventricular nucleus, ARH = Arcuate nucleus, CO Optic chiasma. Dashed zone indicates the area in which all the lesions were found. Usually each rat had at least 50 percent of this zone destroyed.
POA,
delimited
of the der of portion
Table
with
1,
puberty
Lesioned
weights
a lower
animals
was
same
in
was
advanced
Also
body
Nevertheless, was
same.
between
controls
the were
in
controls
in
Figure
the 2
weights to
these
from
was
45
days. a
first
delivery
lesioned was
and
Pituitary lesioned
30
35
IN
40
45
weights Numbers
in in
DAYS
gland and ovarian rats at various ages.
Since
intact
differ-
and
and
lesioned
Twenty-four
by
used
and
animals h after
from
the in
lesion
pituitary
from
lesioned
higher
than
that
of
30. samples
were
plasma
were
rats
during
experimental (Fig.
3).
Plasma
and
in
general
higher
in control
rats
taken
levels
studied
out
fol-
weight
variable
and
ones
weight lesioned
quite
(Fig.
age
and
period
than
in
the
similar the
LH
in entire
levels
were
tended
LH levels
to
be
in lesioned
3). animals 1600,
30
and
days,
lesioned gland
Day
of
ovarian pituitary rats were
control
control
ovarian
FSH
(0800,
as
in
pitui-
days
increased 25 days, lesioned the
was in
pituitary
significantly
blood
When
puberty
were
still
by At
of
trends
controls
difference at 20
thereon
However, was
morning,
control
pituitary
decrease
rats
those
similar
intact
rats. from
From
lowed
rats
lesioned
of
to This present
still
than
control
(see
intact
was
When in
experiments.
ovarian
glands
controls
litterweight
rats
tary
group.
sham
from
compared (P
