Endocrinology Vol. 140, No. 2 595-602
Copyright © 1999 by The Endocrine Society
Changes in Mediobasal Hypothalamic Gonadotropin-Releasing Hormone Messenger Ribonucleic Acid Levels Induced by Mating or Ovariectomy in a Reflex Ovulator, the Ferret1
J. Bakker,
B. S. Rubin and
M. J. Baum
Department of Biology, Boston University (J.B., M.J.B.), Boston,
Massachusetts 02215; and the Department of Anatomy and Cell Biology,
Tufts Medical School (B.S.R.), Boston, Massachusetts 02211
Address all correspondence and requests for reprints to: Dr. Julie Bakker, Department of Biology, Boston University, 5 Cummington Street, Boston, Massachusetts 02215. E-mail: bakker{at}bio.bu.edu
 |
Abstract
|
|---|
The ferret is a reflex-ovulating species in which receipt of an
intromission induces a prolonged (±12 h) preovulatory LH surge in the
estrous female. This LH surge is probably stimulated by a large release
of GnRH from the mediobasal hypothalamus (MBH). In Exp 1 we asked
whether GnRH messenger RNA (mRNA) levels increase in response to mating
so as to replenish the MBH GnRH stores needed to sustain the
preovulatory LH surge. Estrous females were killed 0, 0.25, 0.5, 1, 3,
6, 14, or 24 h after the onset of a 10-min intromission from a
male. Coronal brain sections ranging from the rostral preoptic area
caudally to the posterior hypothalamus were processed for in
situ hybridization using a 35S-labeled oligoprobe
complementary to the human GnRH-coding region. We found no evidence of
increased MBH GnRH mRNA levels during the ferret’s mating-induced
preovulatory LH surge. Instead, the number of GnRH mRNA-expressing
cells dropped significantly in the arcuate region beginning 6 h
after onset of intromission and remained low thereafter. Furthermore,
cellular GnRH mRNA levels decreased in the arcuate region toward the
end of the preovulatory LH surge. In Exp 2 we asked whether ovarian
hormones regulate MBH GnRH mRNA levels in the female ferret.
Ovariectomy of estrous females significantly reduced the number of GnRH
mRNA-expressing cells in the arcuate region. This decrease was probably
not due to the absence of circulating estradiol. Gonadally intact
anestrous females had levels of MBH GnRH mRNA similar to those in
estrous females even though plasma estradiol levels were equally low in
anestrous females and ovariectomized females. Ovarian hormones other
than estradiol may stimulate MBH GnRH mRNA levels in anestrous and
estrous females.
|
Introduction
|
|---|
IN THE FERRET, a reflex ovulator, receipt
of an intromission induces a preovulatory LH surge in the estrous
female (1, 2). This elevation in circulating LH begins around 1.5
h after the onset of intromission, peaks approximately 6 h later,
and is sustained for at least 12 h (2). The preovulatory LH surge
in the female ferret is probably stimulated by a large, sustained
release of GnRH from the mediobasal hypothalamus (MBH) into the
pituitary portal vessels. It was previously found that the in
vitro release from perifused MBH slices and MBH tissue content of
GnRH were significantly reduced in estrous females killed 0.25 h
after receipt of an intromission (3). Also, fewer GnRH-immunoreactive
perikarya were detected in the MBH of ovariectomized, estradiol-primed
female ferrets killed 20 min after receiving mechanical vagino-cervical
stimulation (4). In the vole, another reflex ovulating species, a
similar depletion in hypothalamic GnRH content was found in females 5
min after mating (5). These findings suggest that in these species
mating induces a large release of GnRH from the MBH that initially
depletes GnRH neuronal terminals of peptide. Interestingly, no decrease
in the MBH release of GnRH was observed in estrous female ferrets
killed 1 or 2.6 h after the receipt of an intromission (3),
suggesting that releasable GnRH stores in the MBH are replenished as
early as 1 h after mating. This replenishment could reflect a
mating-induced increase in the biosynthesis of GnRH peptide as a result
of increased GnRH gene expression. In Exp 1, we addressed this question
by comparing GnRH messenger RNA (mRNA) levels in MBH neurons of estrous
female ferrets killed at different times during the course of the
mating-induced preovulatory LH surge.
In spontaneous ovulators such as rat, hamster, sheep, and human,
estrogens exert both positive and negative feedback actions on the
hypothalamus and/or pituitary gland to control LH secretion. In the
ferret, there is only evidence of a negative feedback action of
estrogen (1). Female ferrets in estrus have high levels of circulating
estrogen coupled with low or undetectable levels of LH (6). Ovariectomy
caused a gradual rise in plasma LH in ferrets (6), which was suppressed
by administering estradiol (7). One might expect that the
hypersecretion of LH observed after ovariectomy is driven by increased
GnRH release from the MBH. However, a body of evidence from the rat
(reviewed in Ref. 8) suggests that GnRH release, measured in the MBH
using either in vitro or in vivo methods, is
actually diminished after ovariectomy. Likewise, ovariectomy of estrous
ferrets caused a decrease in the in vitro release and
content of GnRH peptide in the MBH (3). This decrease could reflect a
decrease in the biosynthesis of GnRH peptide in response to a reduction
in GnRH gene expression. In Exp 2, we addressed this question by
comparing GnRH mRNA levels in MBH neurons of ovariectomized female
ferrets as well as gonadally intact estrous and anestrous females. In
both experiments, neuronal GnRH mRNA levels were measured using
isotopic in situ hybridization.
|
Materials and Methods
|
|---|
Animals and experimental design
Adult, gonadally intact, European male and female ferrets in
breeding condition were purchased from Marshall Farms (North Rose, NY).
Subjects were housed individually in modified rabbit cages under a long
day photoperiod (16 h of light, 8 h of darkness; lights on at
0700 h). All ferrets were fed moistened Purina ferret chow
(Ralston Purina Co., St. Louis, MO) once a day. Water was
available ad libitum.
In Exp 1, estrous females received a 10-min intromission from a male in
breeding condition. This mating stimulus reliably provokes a
preovulatory LH surge (2). Mated females were killed 0.25, 0.5, 1, 3,
6, 14, or 24 h after the onset of intromission. Additional estrous
females were taken directly from their home cage and killed (0 h;
unmated controls). All estrous females had fully swollen vulvas, and
all mated females showed high levels of behavioral receptivity. In Exp
2, estrous females were ovariectomized via a single midline incision
and killed 22 days later when plasma LH levels were expected to be high
(7). Additional gonadally intact females in estrus or anestrus were
taken directly from their home cage and killed.
Blood and brain collection
Ferrets were quickly anesthetized using CO2 and
decapitated, and the brains were removed and frozen in powdered dry ice
before being stored at -80 C. Trunk blood was collected in heparinized
tubes. Blood samples were spun down, and plasma was collected and
stored at -20 C before being shipped elsewhere on dry ice for hormone
assays.
Hormone assays
Plasma LH levels were quantified in duplicate in a RIA using the
GDN 15 antiovine LH antiserum (6). The minimum detection level of the
assay was 0.45 ng/ml. The LH assay was performed by Dr. Kathleen Ryan
(Magee-Womens Research Institute, Pittsburgh, PA). Plasma estradiol
levels were measured in duplicate using a double antibody RIA kit
(Diagnostic Products Corp., Los Angeles, CA). The minimum
detection level of the assay was 2 pg/ml. The estradiol assay was
performed by Dr. Geralyn Messerlian Lambert (Womens and Infants
Hospital, Providence, RI).
In situ hybridization for GnRH mRNA
Frozen brains were sectioned coronally at 14 µm using a
cryostat and mounted onto Vectabond-coated slides. Brain sections were
collected beginning rostrally at the level of the organum vasculosum of
the lamina terminalis and extending caudally to the posterior
hypothalamus. Slides were stored in boxes containing desiccant at -80
C until in situ hybridization was performed.
Every fourth brain section was used for in situ
hybridization, which was carried out at the Tufts University Center for
Reproductive Research using a 48-base synthetic oligonucleotide probe
complementary to the GnRH-coding region (bases 102–149) of the human
complementary DNA (9). This oligoprobe has previously been used
successfully in the rat (10) and ferret (11). An initial batch of the
oligoprobe was provided by Dr. Cheryl Sisk of Michigan State University
(East Lansing, MI). Then, additional amounts of the oligoprobe
were synthesized at the Department of Physiology, Tufts Medical School
(Boston, MA). The GnRH oligoprobe was 3'-end labeled by incubation with
[35S]deoxy-ATP (75 pmol; New England Nuclear, Boston, MA)
and terminal deoxynucleotidyl transferase (25 U; Boehringer Mannheim,
Indianapolis, IN) to a specific activity of approximately
106 cpm/µl. The size and the relative purity of the
labeled oligoprobe were determined by gel electrophoresis (Phast
system, Pharmacia, Uppsala, Sweden).
The hybridization protocol was modified slightly from the method used
by Tang et al. (11). Prehybridization treatment consisted of
warming the sections to room temperature, fixing in 4%
paraformaldehyde for 10 min, acetylating with 0.25% acetic anhydride,
dehydrating through a series of ethanols (70%, 80%, 95%, 100%, and
95%), defatting in chloroform, and air-drying at room temperature for
at least 1 h. The 35S-labeled GnRH oligoprobe was
mixed with 2 x SSC, 1 x Denhardt’s solution, 10% dextran
sulfate, 25 µg/ml yeast transfer RNA, and 0.5 mg/ml salmon sperm DNA
to a specific activity of 6 x 103 cpm/µl. The
resulting hybridization solution was heated to 65 C, quenched in ice,
and applied to the sections (20 µl/section). Slides were coverslipped
and placed in humid hybridization chambers overnight at 41 C. After
hybridization, sections were desalted using decreasing concentrations
of SSC (2, 1, and 0.5 x) containing 1 M dithiothreitol
(DTT), followed by a 30-min wash in 0.1 x SSC containing 1
M DTT at 41 C. After a final wash in 0.1 x SSC
containing 1 M DTT at room temperature, sections were
dehydrated through a series of ethanols (50%, 60%, 95%, and 100%)
and air-dried overnight in slide boxes. Slides were dipped into
photographic emulsion (Kodak NTB-3, Eastman Kodak Co.,
Rochester, NY; diluted 1:1 with distilled water) and exposed for 10
days at 4 C. Then slides were developed in Kodak D-19, fixed with Kodak
general purpose fix, counterstained lightly with 0.1% toluidine O
blue, and coverslipped using Permount (Fisher Scientific,
Fairlawn, NJ). Addition of an excess of unlabeled probe to the
hybridization solution completely abolished labeling.
Data analysis
Cell counts. Brain sections from 44 ferrets distributed over
9 in situ hybridization runs were analyzed. Each run
contained brain sections from an unmated estrous female and a subset of
brain sections from different treatment groups. All slides were coded
so that the treatment of the ferret was unknown to the investigator
analyzing the slides. First, all hybridized cells detected in every
section run for in situ hybridization were counted in each
brain region (preoptic area, anterior hypothalamus, arcuate region, and
median eminence). Only cells with more than 5 times the number of
silver grains in the adjacent background were considered labeled. Then
the mean number of GnRH mRNA-expressing cells per section was computed
for each of these 4 brain regions by dividing the total numbers of
hybridized cells by the number of sections included in each brain
region. Then, 4 anatomically matched brain sections (containing at
least 2 hybridized cells/section) from the anterior hypothalamus and
arcuate region and 3 anatomically matched brain sections (containing at
least 1 hybridized cell/section) from the rostral preoptic area and the
median eminence were selected for image analysis. GnRH mRNA-positive
cells were identified by the presence of silver grains overlying a
counterstained cell body.
Image analysis. All GnRH mRNA-positive neurons in the three
or four anatomically matched sections from four brain regions were
digitized for image analysis. Digital images were taken at x1000
magnification using a Zeiss Axioscope (Bellingham, MA) and a Hamamatsu
charge-coupled device video camera together with an 8-bit (256 gray
scale levels) frame grabber board controlled by Bioscan, Inc.’s OPTIMAS image analysis software. A threshold intensity
was set at the level of the underlying counterstained cell body so that
only the silver grains overlying this cell were above this threshold.
In addition, the same set of three GnRH mRNA-expressing cells from one
animal was used as a standard to calibrate the system during each
analysis session. For each hybridized cell, the cell body was
circumscribed manually, and the total hybridization area per cell was
estimated by computing the sum of areas occupied by silver grains. All
hybridized cells found in three or four matched sections per region
were analyzed for each subject. The average hybridization area per cell
was calculated for each brain region for each animal, and these values
were used to determine the group mean and SEM for each
postcoital time point (Exp 1) or endocrine treatment (Exp 2).
Statistical analysis. Because of variability in the
quantitative results, cell numbers and cellular GnRH mRNA values were
compared using nonparametric two-tailed Mann-Whitney U tests. LH and
estradiol levels in plasma were also analyzed using these tests.
|
Results
|
|---|
Distribution of neurons containing GnRH mRNA
Cells expressing GnRH mRNA were recognized as a cluster of silver
grains overlying a counterstained cell. Examples of GnRH mRNA-positive
neurons in the arcuate region from an unmated estrous female (A) and an
ovariectomized female (B) are shown in Fig. 1
. GnRH mRNA-expressing cells were widely
distributed across the MBH (Fig. 2). Only
a few GnRH mRNA cells were detected in the rostral preoptic area and
septal area. Larger numbers of GnRH mRNA cells were found near the base
of the anterior hypothalamus and in the ventral arcuate region, and
only a few GnRH mRNA cells were seen in the median eminence (Fig. 2).
The distribution and number of cells hybridized for GnRH mRNA were
similar to those reported in the male ferret (11) and those found to be
immunoreactive for GnRH protein in ferrets of both sexes (12, 13, 14).
View larger version (167K):
[in this window]
[in a new window]
|
Figure 1. Photomicrographs of cells expressing GnRH mRNA in
the arcuate region (Arc) and the median eminence (Me) of an unmated
estrous female ferret (A) and an ovariectomized female ferret (B). The
high magnification inset in B shows a labeled cell with
silver grains over a counterstained cell body. Arrows
indicate labeled cells.
|
|
View larger version (18K):
[in this window]
[in a new window]
|
Figure 2. Camera lucida drawings of coronal sections through
the rostral preoptic area (+5.0 mm anterior to the interaural line),
anterior hypothalamus (+3.4 mm), arcuate region (+2.6 mm), and median
eminence (+1.9 mm) showing the distribution of GnRH mRNA-containing
neurons ( ) in a
representative, unmated, estrous female ferret. 3V, Third ventricle;
AC, anterior commissure; Arc, arcuate nucleus; Me, median eminence; OC,
optic chiasm; OT, optic tract.
|
|
Effect of mating on GnRH mRNA cell numbers
The mean number of GnRH mRNA-expressing cells in the arcuate
region decreased significantly during the course of the preovulatory LH
surge (Fig. 3). Significantly fewer
hybridized cells were detected in the arcuate region of mated females
killed 6 or 14 h after onset of intromission compared with those
in unmated females (P = 0.03 and P =
0.04, respectively). There was also a trend for cell numbers in the
preoptic area (P = 0.06) in females killed 6 or 24
h after onset of intromission and in the anterior hypothalamus
(P = 0.07) in females killed 6, 14, or 24 h after
the onset of intromission to decline over the course of the
preovulatory LH surge (Fig. 3).
View larger version (37K):
[in this window]
[in a new window]
|
Figure 3. Effect of receipt of an intromission from a male
on the mean (±SEM) number of GnRH mRNA-expressing cells
per brain section. The mean (±SEM) numbers of coronal
brain sections within each region were: preoptic area (POA), 23 ±
1; anterior hypothalamus (AH), 17 ± 1; arcuate region (Arc),
26 ± 1; and median eminence (Me), 26 ± 1. *,
P < 0.05, by two-tailed Mann-Whitney U comparisons
with unmated (0 h) estrous females. The number of subjects per group is
shown above the bars in the top panel.
|
|
Effect of mating on cellular GnRH mRNA levels
Cellular GnRH mRNA levels decreased in the arcuate region over the
course of the preovulatory LH surge (Table 1). Mean cellular levels of GnRH mRNA in
hybridized cells of the arcuate region were significantly lower in
mated estrous females killed 1 or 14 h after the onset of
intromission compared with those in unmated estrous controls (Table 1).
There were no significant mating-induced changes in cellular GnRH mRNA
levels in the preoptic area, anterior hypothalamus, or median eminence
(Table 1).
View this table:
[in this window]
[in a new window]
|
Table 1. Effect of receipt of an intromission from a male on
cellular GnRH mRNA levels in three or four anatomically matched
sections from the mediobasal hypothalamus of estrous female ferrets
|
|
Effect of ovariectomy on GnRH mRNA levels
Ovariectomy significantly decreased the mean number of GnRH
mRNA-expressing cells per section in the arcuate region compared with
those in gonadally intact estrous females (P = 0.03;
Fig. 4). The mean number of GnRH
mRNA-expressing cells did not differ significantly between
ovariectomized females and gonadally intact anestrous females. There
was no effect of ovariectomy on cellular GnRH mRNA levels in any of the
brain regions analyzed (Table 2). In
addition, cellular GnRH mRNA levels did not differ between gonadally
intact estrous and anestrous females.
View larger version (31K):
[in this window]
[in a new window]
|
Figure 4. Effect of ovariectomy on the mean
(±SEM) number of GnRH mRNA-expressing cells per brain
section. The mean (±SEM) numbers of coronal brain sections
within each region were: preoptic area (POA), 19 ± 0; anterior
hypothalamus (AH), 20 ± 1; arcuate region (Arc), 27 ± 1;
and median eminence (Me), 27 ± 1. *, P <
0.05, by two-tailed Mann-Whitney U comparisons with estrous females.
The number of subjects per group is shown above the bars
in the top panel.
|
|
View this table:
[in this window]
[in a new window]
|
Table 2. Effect of ovariectomy on cellular GnRH mRNA levels
in three or four anatomically matched sections from the mediobasal
hypothalamus of female ferrets
|
|
Plasma LH and estradiol
There was evidence of a mating-induced preovulatory LH surge in
estrous females (Fig. 5A). Mean plasma LH
levels were significantly higher in mated females killed 0.25 h
(P = 0.008), 0.5 h (P = 0.004),
1 h (P = 0.02), 14 h (P =
0.01), or 24 h (P = 0.04) after the onset of
intromission than in unmated estrous females (0 h). Ovariectomy
increased plasma LH levels to those seen in mated, estrous females
during the preovulatory LH surge (Fig. 5A). Anestrous females had
plasma LH levels that were intermediate between those of unmated
estrous and ovariectomized females. Both ovariectomized females
(P = 0.004) and anestrous females (P =
0.005) had significantly higher plasma LH levels than unmated estrous
females, whereas ovariectomized females had higher plasma LH levels
than anestrous females (P = 0.009). When the data from
Exp 1 and 2 were combined, no significant correlation was found between
plasma LH levels and the number of GnRH mRNA-expressing cells or
cellular GnRH mRNA levels in any of the four brain regions
analyzed.
View larger version (19K):
[in this window]
[in a new window]
|
Figure 5. LH (A) and estradiol (B) levels in trunk plasma
taken when female ferrets were killed to obtain brains for GnRH mRNA
in situ hybridization. Individual values (solid
circles) and group means (bars) ±
SEM are shown. Anest, Anestrous; ovx, ovariectomized. The
number (N) of subjects per group is given at the bottom
of B. Note that five of eight unmated estrous females had plasma LH
levels that were below the detection limit of the assay (0.45 ng/ml)
and that all ovariectomized females and three of four anestrous females
had plasma estradiol levels that were below the detection limit of the
assay (2 pg/ml).
|
|
Plasma estradiol levels were significantly higher in estrous females
(unmated; 0 h) than in anestrous (P = 0.05) or
ovariectomized females (P = 0.01; Fig. 5B
). Plasma
estradiol levels did not vary significantly among groups of estrous
females killed at different times during the mating-induced
preovulatory LH surge (Fig. 5B). Also, plasma estradiol levels were
equally low in anestrous and ovariectomized females. When data from Exp
1 and 2 were combined, no significant correlation was found between
plasma estradiol levels and the number of GnRH mRNA-expressing cells or
cellular GnRH mRNA levels in any of the four brain regions
analyzed.
|
Discussion
|
|---|
Relationship between GnRH mRNA levels and GnRH release after
mating
We found no evidence of increased GnRH mRNA levels in the MBH
during the ferret’s mating-induced preovulatory LH surge. Instead,
GnRH mRNA levels actually decreased in some MBH regions over the course
of the preovulatory LH surge. In a previous study (3), we reported that
the in vitro release of GnRH from perifused MBH slices was
significantly reduced in estrous female ferrets killed 0.25 h
after the onset of intromission, but was restored to unmated levels in
females killed 1 or 2.6 h after the onset of intromission. These
results suggest that there is a large release of GnRH immediately after
mating that depletes releasable stores of the peptide in MBH nerve
terminals. Replenishment of GnRH stores apparently occurs within 1
h after mating. We hypothesized that an increase in MBH GnRH gene
expression contributes to this replenishment. However, in the present
study MBH GnRH mRNA levels were not elevated during the first hour
after the onset of intromission. The absence of any increase in GnRH
mRNA levels suggests that posttranscriptional events, such as increased
GnRH mRNA translation, increased conversion of the pro-GnRH peptide
into the mature GnRH decapeptide, and/or increased transport of the
peptide to the nerve terminals, contribute to the previously observed
(3) replenishment of GnRH stores in the MBH.
GnRH mRNA levels began to decrease in the MBH during the peak phase of
the preovulatory LH surge. Specifically, the number of GnRH
mRNA-expressing cells was lower in the arcuate region of estrous
females killed 6, 14, or 24 h after the onset of intromission.
This decrease probably reflects a reduction in cellular mRNA content,
so that cells expressing decreased levels of mRNA were no longer
detected with the in situ hybridization method used. Indeed,
cellular levels of GnRH mRNA in the arcuate region decreased
significantly in estrous females killed 14 h after the onset of
intromission. In addition, there was a trend in the preoptic area and
anterior hypothalamus for cellular GnRH mRNA levels and the number of
GnRH mRNA-expressing cells to decline over the course of the
preovulatory LH surge.
The decrease in MBH GnRH mRNA levels during the preovulatory LH surge
may have been caused by reductions in GnRH gene transcription and/or
GnRH mRNA stability. In rats, GnRH mRNA levels appear to be regulated
across the estrous cycle at the level of gene transcription and mRNA
stability (15, 16). For example, GnRH mRNA levels repeatedly exhibit
two significant peaks in cycling rats, one on diestrus and another on
the afternoon of proestrus (15). The increase in GnRH mRNA levels on
diestrus is probably due to an increase in mRNA stability, whereas the
increase on proestrus is accompanied by an increase in gene
transcription (15). However, the decrease in GnRH mRNA levels during
the mating-induced preovulatory LH surge in estrous ferrets is most
likely not the result of changes in ovarian steroid secretion. Plasma
progesterone concentrations do not rise until 5 days after
receipt of an intromission (17), and plasma estradiol concentrations
did not fluctuate significantly during the mating-induced preovulatory
LH surge (present study) and only began to drop 4 days after receipt of
an intromission (17).
In the present study, mean plasma LH levels were significantly elevated
in most groups of mated estrous females, suggesting that they
experienced a mating-induced preovulatory LH surge. This confirms our
previous observations that intromissions that last longer than 2 min
always suffice to induce a preovulatory LH surge in estrous female
ferrets (1, 2, 3). However, there was considerable variability in the
data, with relatively low levels of plasma LH measured in females
killed 1, 3, or 6 h after the onset of intromission. Carroll
et al. (2) demonstrated that the mating-induced preovulatory
LH surge is characterized by increases in pulse frequency and
amplitude. As we took a single trunk blood sample when subjects were
killed for in situ hybridization, we probably measured LH in
both peaks and valleys of these pulses. Wersinger and Baum (14) also
found relatively low plasma LH levels in female ferrets killed 1.5 or
3 h after the onset of a 5-min intromission, suggesting that
synchronized phases of reduced LH secretion occur during the ferret’s
preovulatory LH surge. Even so, Wersinger and Baum (14) found
appreciable numbers of MBH Fos/LHRH double-labeled neurons in all mated
females, including those with low LH levels at the time of death. This
suggests that MBH GnRH neurons were activated in all estrous females
that received an intromission.
Ovarian regulation of GnRH mRNA levels
There appears to be a relationship between levels of GnRH mRNA in
the ferret’s MBH (present study) and the in vitro release
of GnRH peptide from the MBH (3). In estrous female ferrets, GnRH mRNA
(present study) and basal in vitro release and content of
GnRH peptide (3) in the MBH are high. After ovariectomy, GnRH mRNA
levels (present study) and basal in vitro release and
content of GnRH peptide (3) in the MBH are reduced. These data suggest
that ovarian hormones stimulate GnRH gene expression as well as GnRH
release. This stimulatory action of ovarian hormones on GnRH gene
expression and release are in contrast to the negative feedback action
of ovarian hormones on LH release from the pituitary (7, 18). There is
evidence that estrogen promotes GnRH gene transcription in the rat
(16). Estradiol, but not progesterone, treatment increased
levels of GnRH RNA primary transcript in the organum vasculosum of the
lamina terminalis/rostral preoptic area of ovariectomized rats, which
points to an estrogenic induction of GnRH gene transcription (16).
Also, GnRH primary transcript levels were elevated on proestrus in
cycling female rats, a finding consistent with an estrogen-induced
increase in GnRH transcription (15). However, the present study
provides no indication that estradiol is the ovarian hormone that
promotes MBH GnRH mRNA levels in the female ferret. We found equivalent
MBH GnRH mRNA levels in gonadally intact female ferrets that were in
estrus or anestrus even though plasma estradiol levels were
significantly higher in estrous females. Also, there was no positive
correlation between plasma estradiol levels and GnRH mRNA levels in any
brain region studied. The absence of significant differences in GnRH
mRNA levels between estrous and anestrous females suggests that the
reduced function of the hypothalamic-pituitary-gonadal axis during
anestrus might reflect a reduced release of GnRH from the MBH or
reduced sensitivity of the pituitary to GnRH, as opposed to a
down-regulation of MBH GnRH mRNA levels. The reduction in MBH GnRH mRNA
levels after ovariectomy suggests that ovarian hormones other than
estradiol may facilitate MBH GnRH mRNA levels in anestrous and estrous
females.
Species comparisons
Our finding that the mating-induced preovulatory LH surge is
accompanied by progressively decreasing levels of GnRH mRNA is
comparable to earlier findings in sheep. In the ovariectomized ewe, the
estrogen-induced LH surge was associated with decreased levels of GnRH
mRNA in the preoptic area (19, 20). In the rat, however, in those
studies in which changes were reported (reviewed in Ref. 21), increases
in either cellular GnRH mRNA levels or GnRH mRNA cell numbers occurred
in the organum vasculosum of the lamina terminalis/rostral preoptic
area during the estrogen-induced LH surge (10, 15, 22, 23). There is a
species difference in the temporal relationship between the changes
observed in GnRH mRNA levels and the LH surge. In the ferret, in which
the preovulatory LH surge is induced by mating, MBH GnRH mRNA levels
started to decrease during the peak phase of the surge and remained low
until its end. In the sheep, GnRH mRNA levels in the preoptic area
decreased in advance of the onset of the estrogen-induced LH surge
(20). In the rat, GnRH mRNA levels in the organum vasculosum of the
lamina terminalis/rostral preoptic area increased in advance (15) or at
the time of the LH surge (10, 22, 23). These species differences in the
temporal relationship between changes in hypothalamic GnRH mRNA levels
and LH release are one characteristic of the different mechanisms
controlling the preovulatory LH surge in reflex vs.
spontaneous ovulators.
We found that ovariectomy decreased the GnRH mRNA cell number in
the arcuate region of the ferret. A similar decrease in GnRH mRNA
levels has been reported in the rat (24, 25), although this finding is
controversial (reviewed in Ref. 21). Other researchers found no effect
(26) or an increase (27) in GnRH mRNA levels after ovariectomy in
female rats. In the female ferret, estradiol alone appears not to
stimulate MBH GnRH mRNA expression. However, estradiol may play a
permissive role by increasing the sensitivity of MBH GnRH neurons to
activation by somatosensory or olfactory stimuli associated with
mating.
Our finding that anestrous and estrous female ferrets have
equivalent levels of MBH GnRH mRNA agrees with earlier findings in
another long day seasonal breeder, the hamster (28, 29). Male hamsters
chronically exposed to either long or short photoperiods had similar
numbers of hypothalamic GnRH mRNA-expressing neurons. This suggests
that the reduced function of the hypothalamic-pituitary-gonadal axis in
both sexually quiescent male hamsters and anestrous female ferrets is
mediated primarily by reductions in GnRH release or pituitary
sensitivity to GnRH, as opposed to reduced hypothalamic GnRH mRNA
levels.
|
Acknowledgments
|
|---|
We thank Dr. Kathleen D. Ryan at Magee-Womens Research Institute
(Pittsburgh, PA) for generously performing the LH assay, Dr. Geralyn
Messerlian Lambert at Womens and Infants Hospital (Providence, RI) for
generously performing the estradiol assay, Diagnostic Products Corp. (Los Angeles, CA) for providing the reagents for
the estradiol assay, Dr. Cheryl L. Sisk of Michigan State University
(East Lansing, MI) for generously providing an initial batch of
the oligoprobe, and the animal care staff at Boston University (Boston,
MA) for their care of our ferrets. Finally, we thank Dr. Patricia F.
Finn for critical reading of an earlier version of this manuscript.
|
Footnotes
|
|---|
1 This work was supported by Grants HD-21094 and MH-00392 (to M.J.B.)
and P30-HD-28897. 
Received May 19, 1998.
|
References
|
|---|
-
Carroll RS, Erskine MS, Doherty PC, Lundell LA,
Baum MJ 1985 Coital stimuli controlling luteinizing hormone
secretion and ovulation in the female ferret. Biol Reprod 32:925–933[Abstract]
-
Carroll RS, Erskine MS, Baum MJ 1987 Sex
difference in the effect of mating on the pulsatile secretion of
luteinizing hormone in a reflex ovulator, the ferret. Endocrinology 121:1349–1359[Abstract/Free Full Text]
-
Lambert GM, Rubin BS, Baum MJ 1992 Sexual
dimorphism in the effects of mating on the in vitro release of LHRH
from the ferret mediobasal hypothalamus. Physiol Behav 52:809–813[CrossRef][Medline]
-
Bibeau CE, Tobet SA, Anthony ELP, Carroll RS, Baum MJ,
King JC 1991 Vaginocervical stimulation of ferrets induces release
of luteinizing hormone-releasing hormone. J Neuroendocrinol 3:29–36
-
Versi E, Chiapp SA, Fink G, Charlton HM 1982 Effect of copulation on the hypothalamic content of gonadotrophic
hormone-releasing hormone in the vole, Microtus agrestis. J
Reprod Fertil 64:491–494[Abstract/Free Full Text]
-
Ryan KD, Siegel SF, Robinson SL 1985 Influence of
day length and endocrine status on luteinizing hormone secretion in
intact and ovariectomized adult ferrets. Biol Reprod 33:690–697[Abstract]
-
Carroll RS, Baum MJ 1989 Evidence that oestrogen
exerts an equivalent negative feedback action on LH secretion in male
and female ferrets. J Reprod Fertil 86:235–245
-
Kalra SP, Kalra PS 1989 Do testosterone and
estradiol-17ß enforce inhibition or stimulation of luteinizing
hormone-releasing hormone secretion? Biol Reprod 41:559–570[Abstract]
-
Adelman JP, Mason AJ, Hayflick JS, Seeburg PH 1986 Isolation of the gene and hypothalamic cDNA for the common precursor of
gonadotropin-releasing hormone and prolactin release-inhibiting factor
in human and rat. Proc Natl Acad Sci USA 83:179–183[Abstract/Free Full Text]
-
Porkka-Heiskanen T, Urban JH, Turek FW, Levine JE 1994 Gene expression in a subpopulation of luteinizing
hormone-releasing hormone (LHRH) neurons prior to the preovulatory
gonadotropin surge. J Neurosci 14:5548–5558[Abstract]
-
Tang YP, Kashon ML, Sisk CL 1997 Brain
region-specific regulation of luteinizing hormone-releasing hormone
messenger ribonucleic acid in the male ferret: interactions between
pubertal maturation and testosterone. Endocrinology 138:4740–4747[Abstract/Free Full Text]
-
King JC, Anthony ELP 1984 LHRH neurons and their
projections in humans and other mammals: species comparisons. Peptides 5:195–207[CrossRef]
-
Tang YP, Sisk CL 1992 LHRH in the ferret: pubertal
decrease in the number of immunopositive arcuate neurons. Peptides 13:241–247[CrossRef][Medline]
-
Wersinger SR, Baum MJ 1996 The temporal pattern of
mating-induced immediate-early gene product immunoreactivity in LHRH
and non-LHRH neurons of the estrous ferret forebrain. J
Neuroendocrinol 8:345–359[CrossRef][Medline]
-
Gore AC, Roberts JL 1995 Regulation of
Gonadotropin-releasing hormone gene expression in the rat during the
luteinizing hormone surge. Endocrinology 136:889–896
-
Petersen SL, Gardner E, Adelman J, McCrone S 1996 Examination of steroid-induced changes in LHRH gene transcription using
33P- and 35S-labeled probes specific for intron
2. Endocrinology 137:234–239[Abstract]
-
Villars TA, Erskine MS, Lambert GM, Jacobson D, Weaver
DE, Baum MJ 1990 Endocrine correlates of mating-induced reductions
in estrous behavior in an induced ovulator, the ferret. Horm Behav 24:198–214[CrossRef][Medline]
-
Baum MJ, Gerlach JL, Krey LC, McEwen BS 1986 Biochemical and radioautographic analysis of estrogen-inducible
progestin receptors in female ferret brain and pituitary: correlations
with effects of progesterone on sexual behavior and
gonadotropin-releasing hormone-stimulated secretion of luteinizing
hormone. Brain Res 368:296–309[CrossRef][Medline]
-
Dhillon H, Dunn AM, Esquivel E, Hamernik DL, Wise
ME 1997 The estradiol-induced luteinizing hormone surge in the ewe
is not associated with increased gonadotropin-releasing hormone
messenger ribonucleic acid levels. Biol Reprod 57:107–111[Abstract]
-
Harris TG, Robinson JE, Evans NP, Skinner DC, Herbison
AE 1998 Gonadotropin-releasing hormone messenger ribonucleic acid
expression changes before the onset of the estradiol-induced
luteinizing hormone surge in the ewe. Endocrinology 139:57–64[Abstract/Free Full Text]
-
Sagrillo CA, Grattan DR, McCarthy MM, Selmanoff M1996 Hormonal and neurotransmitter regulation of GnRH gene
expression and related reproductive behaviors. Behav Genet 26:241–277
-
Zoeller RT, Young III WS 1988 Changes in cellular
levels of messenger ribonucleic acid encoding gonadotropin-releasing
hormone in the anterior hypothalamus of female rats during the estrous
cycle. Endocrinology 123:1688–1689[Abstract/Free Full Text]
-
Park O-K, Gugneja S, Mayo KE 1990 Gonadotropin-releasing hormone gene expression during the rat estrous
cycle: effects of pentobarbital and ovarian steroids. Endocrinology 127:365–372[Abstract/Free Full Text]
-
Kim K, Lee BJ, Park Y, Cho WK 1989 Progesterone
increases messenger ribonucleic acid (mRNA) encoding luteinizing
hormone-releasing hormone (LHRH) level in the hypothalamus of
ovariectomized estradiol-primed rats. Mol Brain Res 6:151–158[Medline]
-
Roberts JL, Dutlow CM, Jakubowski M, Blum M, Miller
RP 1989 Estradiol stimulates preoptic area-anterior hypothalamic
proGnRH-GAP gene expression in ovariectomized rats. Mol Brain Res 6:127–134[Medline]
-
Kelly MJ, Garret J, Bosch MA, Roselli CE, Douglass J,
Adelman JP, Ronnekleiv OK 1989 Effects of ovariectomy on GnRH
mRNA, proGnRH and GnRH levels in preoptic hypothalamus of the female
rat. Neuroendocrinology 49:88–97[Medline]
-
Toranzo D, Dupont E, Simard J, Labrie C, Couet J,
Labrie F, Pelletier G 1989 Regulation of pro-gonadotropin
releasing hormone gene expression by sex steroids in the brain of male
and female rats. Mol Endocrinol 3:1748–1756[Abstract/Free Full Text]
-
Ronchi E, Krey LC, Pfaff DW 1992 Steady state
analysis of hypothalamic GnRH messenger RNA levels in male Syrian
hamsters: influences of photoperiod and androgen. Neuroendocrinology 55:146–155
-
Porkka-Heiskanen T, Khoshaba N, Scarbrough K, Urban JH,
Vitaterna MH, Levine JE, Turek FW, Horton TH 1997 Rapid
photoperiod-induced increase in detectable GnRH mRNA-containing cells
in Siberian hamster. Am J Physiol 273:R2032–R2039
This article has been cited by other articles:
|
|
|
|
 
J. Bakker, K. R. Kelliher, and M. J. Baum
Mating Induces Gonadotropin-Releasing Hormone Neuronal Activation in Anosmic Female Ferrets
Biol Reprod,
April 1, 2001;
64(4):
1100 - 1105.
[Abstract]
[Full Text]
|
|
|
Top
Abstract
Introduction
