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Laboratory of Molecular Endocrinology, CHUL Research Center and Laval University, Quebec, Canada G1V 4G2
Address all correspondence and requests for reprints to: Dr. Serge Rivest, Laboratory of Molecular Endocrinology, CHUL Research Center and Laval University, 2705 boulevard Laurier, Quebec, Canada G1V 4G2. E-mail: Serge.Rivest{at}crchul.ULaval.Ca
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Introduction |
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The LHRH group of cells in the organum vasculosum of the lamina terminalis (OVLT)/medial preoptic area (MPOA) is believed to be directly involved in controlling the pituitary gonadotropin drive; a majority of LHRH neurons from these areas send their projections to the median eminence (ME) (4, 26). Moreover, a high percentage of LHRH cells localized in this structure that are activated on the afternoon of proestrus (using the immediate-early gene c-fos as an index of neuronal activation) project to the infundibular system (for review, see Ref.27). Interestingly, this group of LHRH neurons seems particularly sensitive to stress-related factors; CRF is capable of perturbing the infundibular LHRH system and reproductive function essentially when microinjected bilaterally into the MPOA (28). Central injection of the cytokine interleukin-1 (IL-1) inhibits spontaneous expression of the nuclear Fos protein occurring in MPOA LHRH neurons during the afternoon of proestrus (29), reduces LHRH gene expression in neurons distributed from the rostral preoptic area/OVLT to the MPOA (30), and interferes with the release of LHRH from the hypothalamus and of LH from the anterior pituitary when administered intracerebroventricularly (29, 31) or directly into the MPOA in proestrous rats (27).
The nature of the challenge seems important in determining the pathways and mechanisms mediating the influence of stress on the LHRH system and reproductive function (27). Indeed, central production of CRF is involved in the inhibition of LHRH and LH release during neurogenic (32) and metabolic (33, 34) stresses, but not after immunogenic and IL-1 challenges (29, 35). Little is known, however, about the ability of stress of many kinds to directly interfere with the biosynthetic machinery of the gene encoding LHRH within the hypothalamus of cycling female rats and the expression of its receptor in the anterior pituitary. In the present study, we have investigated the effects of two different stressors (systemic endotoxin administration and 48-h fasting stress), recognized for their ability to disrupt reproductive function, on LHRH neuronal activity and gene expression in female cycling rats. The intronic-exonic probe technology (22, 36) coupled to the detection of an index of neuronal activation (Fos nuclear spontaneous expression in LHRH cell bodies of the OVLT/MPOA at proestrus) (37, 38) offer a sensitive approach to investigate transcriptional phenomena occurring in LHRH neurons throughout the estrous cycle and under stressful conditions. We also examined the influence of both immune and metabolic challenges on the expression of LHRH-R transcript in the anterior pituitary of cycling female rats.
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Systemic stress
Throughout the day of proestrus and diestrous day 2, rats
received either a single ip injection of lipopolysaccharide (LPS;
L-2880, lot 114H4022, Sigma Chemical Co., St. Louis, MO; 200 µg/100 g
BW) diluted in 300 µl sterile saline (0.9%) or the vehicle solution.
The injections were made at 0900, 1200, and 1500 h, and the rats
were killed 3 h after the ip treatment. The dose of LPS had been
previously tested on a group of female rats and caused a severe immune
activation, with a complete recovery from the bacterial
endotoxin-induced symptomology within the next 24 h. The physical
symptoms observed were shivering, trichiasis, covering themselves, and
immobility, whereas some rats had diarrhea. No mortality occurred
subsequent to this ip treatment, and the rats were conscious and freely
moving at all times throughout the experimental procedure. The times of
death were chosen on the basis of our previous studies, in which we
observed a strong neuronal activation and neuropeptide gene expression
in the hypothalamic nuclei driving the hypothalamic-pituitary-adrenal
axis 3 h after ip treatment with LPS (39, 40). In particular, we
found a pronounced expression of CRF heteronuclear (hn) RNA in the
paraventricular nucleus (PVN) of female rats challenged on the morning
of proestrus (39). The animals were deeply anesthetized via an ip
injection of 0.3 ml of a mixture of ketamine hydrochloride (91 mg/kg)
and xylazine (9 mg/kg) and then rapidly perfused transcardially with
0.9% saline, followed by 4% paraformaldehyde in 0.1 M
borax buffer (pH 9.5; 4 C). Brains and pituitaries were removed from
the skull, postfixed for 2–8 days, and then placed in 10% sucrose in
the solution of 4% paraformaldehyde-borax buffer (pH 9.5) overnight at
4 C. The frozen brains and pituitaries were mounted on a microtome
(Reichert-Jung, Cambridge Instruments Co., Deerfield, IL) and cut into
30-µm coronal and sagittal sections, respectively. The slices were
collected in a cold cryoprotectant solution (0.05 M sodium
phosphate buffer, 30% ethylene glycol, and 20% glycerol) and stored
at -20 C. LHRH transcript (messenger and heteronuclear RNA) in the
OVLT/MPOA and mRNA encoding LHRH-R in the anterior pituitary were
assayed by in situ hybridization histochemistry.
Metabolic stress
After the ovulatory cycle was monitored for at least three or
four consecutive cycles, rats were deprived of food (only water was
given) for approximately 48 h starting on the morning (at
0000 h) of estrus and diestrous day 1 and killed during the day of
diestrous day 2 and proestrus at 1200, 1500, or 1800 h. No
mortality occurred after this treatment, but the rats lost almost
15 g BW/day and were conscious and freely moving at all times
throughout the experimental procedure. Almost 35% of the food-deprived
rats showed a persistent diestrus vaginal monitoring (3 consecutive
days of diestrus-like profile) during the day in which they were
supposed to be in proestrus. However, we cannot rule out the
possibility that the animals normally exhibiting 4-day cycles shifted
into a 5-day cycle under the influence of such challenge. These rats
showing a perturbed cycle were killed together with the intact cycling
rats at corresponding times, but the results were analyzed separately
for fasting with intact cycle and fasting with perturbed cycle
groups.
In situ hybridization histochemistry
Hybridization histochemical localization of each transcript was
carried out in one in six series (every sixth section) of slices
through the brain and the pituitary using 35S-labeled
complementary RNA (cRNA) probes. Protocols for riboprobe synthesis,
hybridization, and autoradiographic localization of mRNA signal were
adapted from the report by Simmons et al. (41). All
solutions were treated with diethylpyrocarbonate (DEPC) and sterilized
to prevent RNA degradation. Tissue sections mounted onto gelatin- and
poly-L-lysine-coated slides were desiccated under vacuum
overnight, fixed in 4% paraformaldehyde for 30 min, and digested by
proteinase K (10 µg/ml in 100 mM Tris HCl, pH 8.0, and 50
mM EDTA at 37 C for 25 min). Thereafter, the brain sections
were rinsed in sterile DEPC water followed by a solution of 0.1
M triethanolamine (pH 8.0), acetylated in 0.25% acetic
anhydride in 0.1 M triethanolamine, and dehydrated through
graded concentrations of alcohol (50%, 70%, 95%, and 100%). After
vacuum drying for a minimum of 2 h, 90 µl hybridization mixture
(107 cpm/ml) were spotted on each slide, sealed under a
coverslip, and incubated at 60 C overnight (
15–20 h) in a slide
warmer. Coverslips were then removed, and the slides were rinsed in
4 x standard saline citrate (SSC) at room temperature. Sections
were digested by ribonuclease A (20 µg/ml, 37 C, 30 min), rinsed in
descending concentrations of SSC (2, 1, and 0.5 x), washed in 0.1
x SSC for 30 min at 60 C (1 x SSC = 0.15 M NaCl
and 15 mM trisodium citrate buffer, pH 7.0), and dehydrated
through graded concentrations of alcohol. After being dried for 2
h under vacuum, the sections were exposed at 4 C to x-ray film (Eastman
Kodak, Rochester, NY) for 12–36 h (depending on the probe), defatted
in xylene, and dipped in NTB-2 nuclear emulsion (Kodak; diluted 1:1
with distilled water). Slides were exposed for a maximum of 1 week
(LHRH mRNA, 2 days; LHRH hn/mRNA, 5 days; LHRH-R mRNA, 7 days),
developed in D19 developer (Kodak) for 3.5 min at 14–15 C, and fixed
in rapid fixer (Kodak) for 5 min. Thereafter, tissues were rinsed in
running distilled water for 1–2 h, counterstained with thionin
(0.25%), dehydrated through graded concentrations of alcohol, cleared
in xylene, and coverslipped with DPX mountant for microscope (BDH Lab.
Supplies, Poole, England).
cRNA probe synthesis and preparation
LHRH cRNA probe was generated from the rat LHRH complementary
DNA (cDNA; Dr. J. P. Adelman, Department of Cell Biology and Vollum
Institute for Advanced Biomedical Research, Oregon Health Sciences
University, Portland, OR), subcloned into 6ST7, and linearized with
HindIII. A LHRH genomic fragment covering 506 bp of the
intron B-exon 3-intron C junction (Drs. A. Gore and J. L. Roberts,
Research Center in Neurobiology, Mount Sinai, NY) was subcloned in
pBluescript+ (Stratagene, La Jolla, CA). This probe yielded
a band (506 bp) corresponding to the intron B-exon 3-intron C junction
[used as an index of primary transcript (LHRH hn/mRNA)] (36) and was
linearized with EcoRI and HindIII (Pharmacia
Biotech, Montreal, Canada) for antisense and sense, respectively.
LHRH-R cRNA probe (1.2 kilobases) was obtained by PCR, subcloned into
pcDNAI (Dr. M. Perrin and W. Vale, The Salk Institute, La Jolla, CA),
and linearized with BamHI and EcoRI for antisense
and sense, respectively.
Radioactive antisense cRNA copies were synthesized by incubation of 250
ng linearized plasmid in 6 mM MgCl2, 30–40
mM Tris (pH 7.5), 2 mM spermidine, 10
mM dithiothreitol, 0.2 mM ATP/GTP/CTP,
[
-35S]UTP, 40 U RNAsin (Promega, Madison, WI), and 20
U T7 (LHRH mRNA), T3 (LHRH hn/mRNA), or SP6 (LHRH-R mRNA) RNA
polymerase for 60 min at 37 C. Unincorporated nucleotides were removed
using the ammonium-acetate precipitation method; 100 µl
deoxyribonuclease solution (1 µl deoxyribonuclease, 5 µl 5 mg/ml
transfer RNA, and 94 µl 10 mM Tris-10 mM
MgCl2) were added, and 10 min later an extraction was
accomplished using a phenol-chloroform solution. The cRNA was
precipitated with 80 µl 5 M ammonium acetate and 500 µl
100% ethanol for 20 min on dry ice. The pellet was washed with 500
µl ethanol, dried, and resuspended in 100 µl 10 mM
Tris-1 mM EDTA (pH 8.0). A concentration of 107
cpm probe was mixed into 1 ml hybridization solution [500 µl
formamide, 60 µl 5 M NaCl, 10 µl 1 M Tris
(pH 8.0), 2 µl 0.5 M EDTA (pH 8.0), 50 µl 20 x
Denhart’s solution (Ficoll 1%, polyvinylpyrrolidone 1%, BSA fraction
V 1%), 200 µl 50% dextran sulfate, 50 µl 10 mg/ml transfer RNA,
and 10 µl 1 M dithiothreitol (118 µl DEPC water -
volume of probe used)]. This solution was mixed and heated for 5 min
at 65 C before being spotted onto slides. Radioactive sense (control)
cRNA copies were also prepared to verify the specificity of each probe.
Hybridization with these probes did not reveal any positive signal in
the brains of control and stressed rats.
Immunocytochemistry
Immunocytochemistry for Fos-immunoreactive (-ir) neurons was
combined with immunocytochemistry for LHRH-ir neurons to determine
whether LHRH-ir cells stained for Fos protein in the OVLT/MPOA of
endotoxin-challenged and food-deprived rats during the afternoon of
proestrus. Every sixth tissue slice was processed by using the
avidin-biotin amplification bridge method with peroxidase as a
substrate. Briefly, slices were washed in 0.05 M potassium
PBS (KPBS) and incubated at 4 C with Fos antibody mixed in KPBS, 0.4%
Triton X-100, and 1% BSA (fraction V, Sigma Chemical Co.). Rabbit
antihuman/rat Fos serum (Ab-5, rabbit polyclonal IgG, catalog no. PC38
Oncogene Science, Uniondale, NY) was used at a concentration of 1:5000.
Approximately 40 h after incubation at 4 C with the primary
antibody, the brain slices were rinsed in KPBS and incubated with a
mixture of KPBS, Triton-X, and biotinylated goat antirabbit IgG (1:1500
dilution; Vector Laboratories, Burlingame, CA) for 90 min. Sections
were then rinsed with KPBS and incubated at room temperature for 60 min
with an avidin-biotin-peroxidase complex (Vectastain ABC elite kit,
Vector Laboratories). After several rinses in KPBS and
acetate-imidazole buffer (175 mM Na acetate and 10
mM imidazole), brain slices were reacted in a mixture
containing acetate-imidazole buffer, nickel (II)
sulfate·6H2O (100 mM) and the chromagen
3,3'-diaminobenzidine tetrahydrochloride (DAB; 0.03%) along with
0.003% hydrogen peroxide (H2O2).
The staining of LHRH neurons was performed by means of a dual immunoperoxidase method. Immediately after Fos protein staining, the tissues were rinsed in acetate-imidazol buffer followed by KPBS, then incubated with the LHRH antibody (LR-1, a generous gift from Dr. Robert Benoit, Montreal General Hospital, McGill University, Montreal, Canada) for 48 h at 4 C. Rabbit anti-LHRH was used at a concentration of 1:10,000. After incubation with the first antibody, tissues were rinsed, and the avidin-biotin bridge was formed as described above. LHRH structures were stained in Tris-imidazole buffer containing the chromagen DAB (0.05%) and 0.003% H2O2. Thereafter, tissues were rinsed in KPBS, mounted onto subbed glass slides, dehydrated through graded concentrations of alcohol, cleared in xylene, and coverslipped with DPX. Although the presence of Fos protein was evident as a blue-black reaction product in cell nuclei, LHRH-ir within the cell cytoplasm was stained in yellow-gold.
Combination of immunocytochemistry and in situ hybridization
histochemistry
Immunocytochemistry (Fos-ir neurons) was combined with the
in situ hybridization histochemistry protocol (LHRH mRNA) to
determine whether LHRH-expressing cells stained for Fos protein in the
OVLT/MPOA of control and challenged animals. Every sixth tissue slice
was processed by using the avidin-biotin amplification bridge method
with peroxidase as a substrate. Briefly, slices were washed in sterile
DEPC-treated 0.05 M potassium PBS (KPBS) and incubated at 4
C with Fos antibody mixed in sterile KPBS, 0.4% Triton X-100, 1% BSA,
0.25% heparin sodium salt USP (ICN Biomedicals, Aurora, OH), and 1%
BSA (fraction V, Sigma Chemical Co.). Rabbit antihuman/rat Fos serum
was used at a concentration of 1:5000. Approximately 24 h after
incubation at 4 C with the primary antibody, the brain slices were
rinsed in sterile KPBS and incubated with a mixture of KPBS, Triton X,
BSA, heparin, and biotinylated goat antirabbit IgG (1:1500 dilution;
Vector Laboratories) for 90 min. Sections were then rinsed with KPBS
and incubated at room temperature for 60 min with an
avidin-biotin-peroxidase complex (Vectastain ABC elite kit, Vector
Laboratories). The peroxidase complex was amplified by means of 10-min
incubation with a 70-nM solution of biotin
[sulfosuccinimydyl 6-(biotinamido)hexanoate; no. 21335, Pierce
Chemical Co., Rockford, IL]-tyramine HCl (4-hydroxyphenethylamine
hydrochloride, Sigma T-2879)-H2O2 (0.01%),
followed by a second incubation of 30 min with the ABC elite solution.
After several rinses in sterile KPBS, the brain slices were reacted in
a mixture containing sterile KPBS, the chromagen DAB (0.05%), and
H2O2 (0.003%).
Thereafter, tissues were rinsed in sterile KPBS, mounted onto poly-L-lysine-coated slides, desiccated under vacuum overnight, fixed in 4% paraformaldehyde for 30 min, and digested by proteinase K [10 µg/ml in 100 mM Tris-HCl (pH 8.0) and 50 mM EDTA (pH 8.0)] at 37 C for 25 min). Prehybridization, hybridization, and posthybridization steps were performed as described above with the difference of dehydration (50%, 70%, 95%, and 100% alcohol), which was shortened to avoid decoloration of Fos protein (brown staining). After drying for 2 h under the vacuum, sections were exposed at 4 C to x-ray film (Kodak) overnight, defatted in xylene, and dipped in NTB2 nuclear emulsion (Kodak; diluted 1:1 with distilled water). Slides were exposed for 2 days, developed in D19 developer (Kodak) for 3.5 min at 15 C, and fixed in rapid fixer (Kodak) for 5 min. Thereafter, tissues were rinsed in running distilled water for 1–2 h, rapidly dehydrated through graded concentrations of alcohol, cleared in xylene, and coverslipped with DPX. The presence of LHRH mRNA was evident as silver grains in perikarya, and Fos-ir within the cell nuclei was stained in brown.
Quantitative analysis
LHRH-expressing neurons were identified using bright- and
darkfield microscopy to localize grain cluster over cell bodies, and
only cells that expressed at least 5 times the background were included
in the calculation. The number of LHRH neurons was counted in 1 in 6
series (every sixth section) of slices, and the estimated total was
obtained by multiplying the number by 6. An average of 4 sections were
analyzed per animal. Semiquantitative analysis of hybridization signals
for LHRH primary transcript was carried out in nuclear emulsion-dipped
slides over the confines of cells within the rostro-medial OVLT/MPOA
using an Olympus Optical System (BX-50, BMax, Olympus Corp., Melville,
NY) coupled to a Macintosh computer (PowerPC 7100/66) and Image
software (version 1.59 non-FPU, W. Rasband, NIH, Bethesda, MD). The
optical density (OD) of the hybridization signal was measured under
darkfield illumination at a magnification of x100, providing arbitrary
units of signal intensity. Sections from the experimental and control
animals were matched as closely as possible for rostrocaudal level.
Every neuron was digitized and subjected to densitometric analysis,
yielding measurements of integrated OD. The OD of each cell was then
corrected for the average background signal, which was determined by
sampling cells immediately outside the cell of interest (42). The total
OD for all the LHRH-positive cells of the rostro-medial OVLT/MPOA area
was also calculated by the addition of each digitized neuron
(OD/neuron) for the entire group of LHRH-expressing cells located in
the above-mentioned region. The OD of the hybridization signal for the
adenohypophysis LHRH-R was measured under darkfield illumination at a
magnification of x10, providing arbitrary units of signal intensity.
Three areas of each anterior pituitary section were digitized and
subjected to densitometric analysis, yielding measurements of
integrated OD. The average OD number per rat included 9–12
measurements (3 values for each pituitary section; 3–4
sections/animal). On the other hand, both the number of LHRH-ir cells
and the number of LHRH mRNA-expressing neurons exhibiting Fos-ir in
their nuclei were determined under high magnification microscopic
evaluation. The percentage of the total number of LHRH-positive cells
expressing the immediate-early gene within the OVLT/MPOA was calculated
and is represented as the average number of double-labeled neurons for
a 1 in 6 series of brain sections.
Statistical analysis
The results shown in Figs. 5
and 7 were analyzed by a three-way
(3 x 2 x 3) ANOVA, and post-hoc comparisons were
made using a Bonferroni-Dunn test (StatView 4.01, Abacus Concepts,
Inc., Berkeley, CA). Factors were identified as treatment, which was
composed of three levels (LPS, fasting, and control); phase, including
two levels (proestrus and diestrous day 2); and time, which was divided
into three levels (T-12, T-15, and T-18). The data presented in Fig. 2
were analyzed by a two-way (3 x 3) ANOVA followed by a
Bonferroni-Dunn post-hoc test.
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Results |
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illustrates a representative example of
LHRH-expressing cells (left panel) and LHRH-ir neurons
(right panel) displaying Fos-ir nucleus during the afternoon
of proestrus. Although the presence of the blue-black nucleus in the
right panel localized within LHRH-ir cytoplasm is clearly
distinguishable, Fos-ir nucleus surrounded by cytoplasmic silver grains
(left panel) is rather faint because of the yellow-gold
color of the DAB chromagen not intensified with the nickel sulfate. In
fact, when immunocytochemistry is combined with in situ
hybridization, nickel-sulfate intensification cannot be used, as it
vanishes almost entirely during the in situ procedure. As
shown in Fig. 2
(top panel), almost 40% of
the LHRH mRNA-labeled cells spontaneously expressed Fos protein in
their nuclei during the afternoon of proestrus. Interestingly, 3 h
after endotoxin administration, the number of LHRH neurons expressing
Fos was significantly decreased, and a significant interaction between
stress and time was found on the afternoon of proestrus. Expression of
Fos within the LHRH-ir perikarya of the OVLT/MPOA was also altered by
LPS injection, and this phenomenon was significantly more pronounced in
the late afternoon of proestrus (Fig. 2, bottom panel). On
the other hand, a 48-h fasting stress had a modest (if any) influence
on the percentage of LHRH-expressing and LHRH-ir neurons exhibiting
nuclear Fos protein in intact cycling animals. However, food
deprivation was able to disrupt estrous cyclicity in 35% of the rats
used in the study. This group of animals exhibited a persistent
diestrous monitoring by vaginal smear, and as expected in rats that are
not in proestrus, Fos protein was undetectable within the nucleus of
LHRH-positive neurons (data not shown).
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summarizes the in situ
hybridization data obtained via the intronic probe technology used to
detect LHRH primary transcript (hn/mRNA) in the OVLT/MPOA of cycling
female rats challenged by LPS administration (immune stress) and at
least 48 h of fasting (metabolic stress). The relevance of using
this type of probe to investigate the effects of various stressors on
LHRH gene expression is justified by the lack of sensitivity of exonic
probe to detect small changes in specific cellular population. Indeed,
exonic probe technology is a powerful tool to investigate the
distribution and organization of the LHRH system because of the very
strong signal generally detected by in situ hybridization
(Fig. 3, left panels). This strong signal
observed in most of the LHRH neurons of the OVLT/MPOA rends evaluation
of mRNA levels barely possible, and small changes in the expression of
that transcript cannot be detected. However, a significant reduction of
the number of silver grains can be observed in LHRH cells using
intronic probe technology (Fig. 3, right panels) compared to
that using a LHRH exonic cRNA probe (Fig. 3, left panels).
Significant changes in the levels of LHRH primary transcript have
recently been reported using this probe (22), and this tool allowed us
to quantify the hybridization signal in LHRH neurons distributed in the
OVLT/MPOA of stressed cycling animals.
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However, a significant interaction between the phase and the time of
the estrous cycle was found; a modest, but significant, increase in the
number of LHRH-expressing neurons was detected at 1800 h on
diestrous day 2. The hybridization signal for all LHRH-positive neurons
located in the OVLT/MPOA was higher at 1500 h on proestrus in
control animals, although none of the challenges significantly altered
this variable (Table 1, middle column). The average OD per
cell expressing LHRH primary transcript was also higher in female rats
killed at 1500 h on the day of proestrus, an event significantly
attenuated by endotoxin administration. Figure 4 shows
representative examples of this phenomenon for the LHRH cell bodies
forming the inverted V surrounding the OVLT. Interestingly, compared to
their corresponding control groups at each phase of the estrous cycle,
LPS treatment did not affect the transcription of LHRH at times other
than 1500 h on proestrus (Fig. 5). Similarly, food
deprivation did not significantly alter the average OD per cell, apart
from the slight decrease observed during the afternoon of proestrus at
1500 h. However, when the estrous cycle was disrupted, there was a
significant alteration in LHRH transcription (see below).
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35%) was, however, killed together with the other rats
showing clear proestrous smearing; data, analyzed separately,
demonstrated that 1) the number of total LHRH-positive neurons for LHRH
hn/mRNA was significantly decreased compared to that in control rats at
1500 h on proestrus (390 ± 27 vs. 498 ±
12); 2) both the total OD in the OVLT/MPOA and the average OD per
neuron of hybridization signal were significantly reduced compared to
those in control animals in the late afternoon of proestrus (4125
± 53 vs. 8918 ± 295 and 67 ± 4 vs.
128 ± 4.6 at 1500 h and 4949 ± 867 vs.
7283 ± 863 and 56 ± 19 vs. 106 ± 7.3 at
1800 h, respectively). As shown in Table 1, the same tendency was
noticed when rats displaying disturbed cycles were compared to normal
diestrous rats. In addition, the LHRH-R hybridization signal in the
anterior pituitary of food-deprived female rats showing perturbed
cycles was strongly down-regulated at both at 1500 and 1800 h
compared to that in control proestrus rats (42 ± 0.2 and
48.5 ± 0.5 vs. 22.6 ± 0.9 and 21.4 ± 0.9,
respectively).
mRNA encoding the receptor for LHRH in the female rat anterior
pituitary
Figure 6 depicts a representative example of LHRH-R
mRNA expression in the pituitary of control and LPS-treated cycling
female rats on the afternoon of proestrus. A strong positive signal was
observed in the adenohypophysis of control rats, and no significant
differences were observed according to the phase of the estrous cycle
(Fig. 7). On the contrary, the hybridization signal for
LHRH-R transcript was strongly inhibited in the anterior pituitary of
LPS-injected rats, and this effect seemed permanent throughout the
estrous cycle (Fig. 7). A more variable pattern of LHRH-R gene
expression was found after food deprivation; LHRH-R was significantly
down-regulated only during particular phases of the estrous cycle,
including 1800 h on proestrus, 1500 h on diestrous day 2, and
1800 h on diestrous day 2.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The activity of the LHRH system during the ovulatory cycle is the result of an intriguing puzzle of neuromolecular steps aimed at regulation of the pituitary LH surge in a critical period during the proestrus day. A complex network of neurochemical pathways temporally integrates the amount of central and peripheral information leading to the secretory events in LHRH neurons (7). Although converging data have largely expanded our understanding of LHRH system behavior at the time of ovulation, what really takes place in hypothalamic LHRH neurons in terms of both gene expression and protein products is far from clear. Moreover, the mechanisms involved in the ability of particular stressful conditions to affect LHRH neuronal activity and the biosynthetic machinery of the decapeptide remain poorly described. However, the possibility of examining this neuroendocrine system by means of exonic and intronic probe technologies (36) coupled to the detection of spontaneous nuclear-early gene expression in OVLT/MPOA neurons during the afternoon of proestrus (37, 38, 43) provided new tools to clarify many aspects of LHRH transcription during basal and perturbed conditions. Several groups have reported low LHRH gene expression during the morning of proestrus compared to that in the afternoon (9, 10, 11). Levine and co-workers observed fluctuations in the number of cells expressing LHRH mRNA in specific regions of the basal forebrain during the periovulatory period (12), whereas Silverman and Witkin did not observe any regional differences and found that after the preovulatory surge, all LHRH neurons were synchronized to start neosynthesis of the peptide together (13). Our results did not show a clear increase in the number of LHRH-expressing neurons during the periovulatory period, although an elevation of LHRH primary transcript was detected at 1500 h on proestrus. These results are in agreement with the recent studies suggesting that transcriptional events take place in LHRH neurons selectively during the afternoon of proestrus (22).
Interestingly, endotoxin injection significantly prevents the spontaneous expression of Fos protein in LHRH-expressing neurons and the increase in LHRH primary transcript during the afternoon of proestrus. These data support the concept that the biosynthetic machinery of the gene encoding LHRH is activated during the preovulatory LH surge and that immune challenge can interfere with LHRH neuronal activity at the transcriptional level. The exact participation of the immediate-early gene c-fos in these events remains highly hypothetical. Parallel alteration of both Fos and LHRH gene expression was detected in the group of neurons analyzed in the present study. Because c-fos is a transcription factor generally recognized to activate genes when forming a heterodimer with members of the c-jun family (44) and because an activating protein-1 (AP-1) DNA-binding site is present on LHRH promoter (45), it is tempting to speculate that both phenomena (inhibition of Fos and LHRH transcription) are related. However, recent studies have provided the evidence that AP-1 is involved in repressing, rather than stimulating, LHRH gene transcription, at least in the GT1–7 neuronal cell line (45).
Although the inhibitory influence of LPS on OVLT/MPOA LHRH neurons was quite convincing during the afternoon of proestrus, the effect of food deprivation seemed more variable and associated with reproductive cyclicity. The detrimental influence of acute and/or chronic food restriction on reproductive performance have been reported in females of several mammalian species (46, 47, 48). Food-deprived rats have been shown to exhibit decreased levels of gonadotropins (49), normal LHRH release from the ME (50), and a preserved LH response to exogenous LHRH (49). On the other hand, a decreased number of neurons expressing LHRH mRNA was found in male rats after a 2- to 3-day fasting stress (51), whereas the inhibition of LH secretion in food-restricted lambs was not associated with decreased LHRH biosynthesis (52). In the present study, food deprivation did not cause significant changes in LHRH neuronal activity and gene transcription in the OVLT/MPOA in intact cycling animals, but a significant decrease in the levels of LHRH primary transcript was found in rats exhibiting an impairment of reproductive cyclicity. It is likely that the metabolic signal differs between animals, and the perception of the challenge was more stressful in some animals. The metabolic and neurogenic stressful conditions may together deeply perturb the LHRH neuronal system and the estrous cycle. However, 65% of rats maintained their cycle intact, and little alteration of the neurons controlling reproductive function was detected. Whether these rats were less sensitive to nutritional signal or more resistant to stress remains an open question. The possibility that body weight plays a role in these differential effects of fasting on the estrous cycle was also taken into consideration, but no correlation between the body weight and disruption of cyclicity was observed (data not shown).
Despite the recognized existence of a powerful interaction between
stress-related factors and the LHRH neuronal system, we still know
little of the pathways through which the hypothalamic network
transduces the effects of various kinds of stress on reproductive
performance. It was tempting to speculate that the PVN of the
hypothalamus had a leading role in such interplay, because this
endocrine nucleus is the principal site of neuroendocrine CRF, which
drives the activity of the corticotroph axis during emergency
situations (53, 54). Increased central levels of CRF have also been
shown to be involved in the antireproductive effect of neurogenic and
metabolic stress (for review, see Ref.27). Interesting,
fasting-induced suppression of LH release in female rats evolves
central CRF and ascending catecholaminergic projections to the PVN; the
injection of a specific CRF antagonist and/or a tyrosine hydroxylase
inhibitor directly into the PVN is capable of restoring pulsatile LH
release in ovariectomized estradiol-treated rats (33). In contrast,
complete bilateral electrolytic lesions of the PVN failed to prevent
the decrease in LH release caused by both neurogenic and IL-1
challenges in castrated male rats (55). Moreover, administration in the
lateral ventricles of different types and doses of CRF antagonists did
not modify the ability of IL-1 and IL-1ß to inhibit LHRH neuronal
activity and plasma LH levels in male and female rats (29). In
addition, peripheral administration of endotoxin, which significantly
increases the endogenous release of peripheral and central cytokines
(56, 57), strongly decreases circulating LH levels (58), a phenomenon
partially reversed by antibodies against IL-1ß (59), but not anti-CRF
(60).
Neurons expressing LHRH in the MPOA/OVLT seem to be the major target of many stress-related factors; infusion of CRF bilaterally into the MPOA significantly inhibits LHRH release into the ME, whereas the same procedure directly into other hypothalamic nuclei or the infundibular system does not cause notable changes in hypothalamic LHRH secretion during proestrus (28). Microinfusion of IL-1ß bilaterally into the MPOA also reduces hypothalamic LHRH release and prevents the spontaneous expression of Fos within LHRH cell nuclei during the afternoon of proestrus (27). However, the physiological relevance of these results remains largely unknown. In fact, LHRH neurons do not seem to express any of the CRF and IL-1 type 1 and 2 receptors (Nappi, R. E., and S. Rivest, unpublished data). Moreover, the MPOA displays a very low to barely detectable signal for both CRF receptor transcripts in intact cycling and stressed female rats (61). Similarly, the gene encoding type I IL-1 receptor has been shown to be expressed in few discrete cell groups, and no specific labeling for type 1 IL-1R mRNA was detected over neurons of the MPOA (62).
Systemic LPS administration induces a profound down-regulation of LHRH-R mRNA expression in the anterior pituitary regardless of the phase of the estrous cycle. This suggests that activation of the acute-phase response is also able to interfere with the HPG axis at the pituitary level independently of the circulating gonadal steroid concentrations. Interestingly, systemic administration of IL-1ß does not affect LH secretion (63), whereas peripheral administration of endotoxin strongly decreases circulating LH levels (31, 59). It is, therefore, likely that a complex cascade of cytokines produced during the acute-phase response is required to decrease the activity of the HPG axis. Concomitant actions of several circulating factors probably operate at the level LHRH neuronal activity and/or the pituitary gland (as paracrine activity) to mediate the inhibition of reproductive function in response to immune challenge. The effect of food deprivation on LHRH-R gene expression in the anterior pituitary was more variable and occurred at specific periods of the estrous cycle. We cannot exclude the possibility that sex steroids play a role in the ability of metabolic challenges to alter the expression of LHRH-R in the adenohypophysis, although the pattern of inhibition does not follow the marked hormonal changes generally observed during the cycle. It is of interest to note that in cycling female rats that did not have a regular estrous cycle during food deprivation, a significant down-regulation of pituitary LHRH-R mRNA was found in all animals.
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Acknowledgments |
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Footnotes |
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2 While at the CHUL Research Center, R.E.N. was a postdoctoral fellow
supported in part by IRCCS Policlinico S. Matteo, University of Pavia,
and by Consiglio Nazionale delle Ricerche (Pos. 121.16479), Italy.
Permanent address: Department of Obstetrics and Gynecology, University
of Pavia, Piazzale Golgi 2, Pavia, Italy.
3 Medical Research Council Scholar of Canada.
Received October 25, 1996.
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and ß in endotoxin-induced suppression of plasma
gonadotropin levels in rats. Endocrinology 130:3307–3313
acts at the level of the brain and the gonads to interfere with
gonadotropin and sex steroid secretion. Endocrinology 124:2105–2109This article has been cited by other articles:
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