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-Containing Neurons in the Preoptic Area of the Ewe Depends on Reproductive Status1
Department of Physiology, West Virginia University Health Sciences Center (I.S., B.A., R.L.G.), Morgantown, West Virginia 26506-9229; and the Department of Cell Biology, Neurobiology, and Anatomy, University of Cincinnati College of Medicine (H.T.J., M.N.L.), Cincinnati, Ohio 45267-0521
Address all correspondence and requests for reprints to: Dr. Robert L. Goodman, Department of Physiology, West Virginia University Health Sciences Center, P.O. Box 9229, Morgantown, West Virginia 26506-9229. E-mail: rgoodman{at}wvu.edu
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Abstract |
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(ER). This apparent discrepancy raises the possibility
that at least one other neural system is also involved in mediating
E2 inhibition. The purpose of this study was to determine
whether ER-containing neurons are activated by the negative feedback
action of E2 in anestrus.
In Exp 1, we examined the effects of E2 on expression of
the immediate early gene products, Fos and Fos-related antigens, in
ER-positive cells in anestrous ewes. ER and Fos/ Fos-related
antigens were colocalized using a dual immunofluorescence procedure in
sections throughout the hypothalamus from ovariectomized and
E2-treated ovariectomized anestrous ewes. A low dose
E2 treatment that inhibited LH pulse frequency and induced
Fos in A15 dopaminergic neurons in a previous study significantly
increased the percentage of ER-containing neurons expressing Fos
(17.8% vs. 1.7%) in the medial preoptic area, but not
in other hypothalamic areas. In Exp 2, we determined whether there was
a seasonal difference in the effect of E2 on Fos/ER
colocalization in this region. E2 treatment produced a
3-fold increase in the percentage of ER-positive cells expressing
Fos (15.1% vs. 3.4%) in anestrus, but failed to
increase ER/Fos colocalization (1.8% vs. 3.5%)
during the breeding season. These data raise the possibility that a
subset of ER-containing neurons in the medial preoptic area plays a
role in the seasonal change in response to E2 negative
feedback in the ewe.
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Introduction |
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The results of pharmacological studies have implicated dopamine (DA) as an important neurotransmitter mediating E2 negative feedback in anestrus (6, 7). Lesion experiments (8, 9) have identified a specific group of dopaminergic (DA) cells (the A15 group) in the retrochiasmatic area of the ovine hypothalamus that are important for E2 negative feedback during anestrus. Injection of the neurotoxin 6-hydroxydopamine into the A15 area partially decreased the ability of E2 to inhibit LH pulse frequency in anestrous ewes (8). Radiofrequency lesions in this area also decreased both the inhibitory effects of E2 and the stimulatory effects of the dopamine antagonist, pimozide, on LH pulse frequency during anestrus (9). However, these lesions failed to affect E2 inhibition of LH pulse amplitude during the breeding season. Therefore, it has been proposed that seasonal changes in the activity of these cells play a major role in controlling the annual reproductive cycle of the ewe.
E2 also appears to stimulate the activity of these A15 cells during anestrus, as indicated by increased tyrosine hydroxylase bioactivity in vivo (10) and increased multiunit electrical activity (11) in this region. Both effects of E2 were associated with or preceded an E2-induced reduction in LH secretion. Using the early immediate gene product, Fos, as an index of neuronal activation, we have confirmed that E2 stimulates the activity of these DA perikarya. Treatment of ovariectomized ewes with E2 significantly increased the percentage of A15 DA neurons expressing Fos in anestrus, whereas the same treatment in the breeding season had no effect (12).
Actions of E2 in the adult brain are thought to
be mediated by intracellular binding of this hormone to nuclear
estrogen receptors (ER) (13). However, DA cells in the A15 area do not
appear to contain ER (14, 15). There are thus two possible
mechanisms by which E2 could stimulate these
neurons. First, they may contain ERß (16, 17). Alternately,
E2 could act on other neurons that contain ER and
that project to and stimulate A15 DA cells. These studies examined the
latter possibility.
We hypothesized that a population of ER-containing neurons would be
activated by an E2 treatment that induced Fos in
A15 DA neurons in anestrus. Two experiments were performed to test this
hypothesis. In the first experiment, we examined hypothalamic areas
known to contain ER for Fos/ER colocalization, using tissue from
the same animals in which we previously observed tyrosine
hydoxylase/Fos colocalization in the A15 area (12). In the second
experiment, we tested whether the ability of E2
to stimulate a specific subset of ER-containing cells varied
seasonally.
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Materials and Methods |
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Bilateral ovariectomies were performed via midventral laparotomy, using sterile procedures and pentobarbital anesthesia. Serial blood samples were collected via jugular venipuncture at 12-min intervals to monitor LH pulse patterns and verify the inhibitory effects of E2 treatments on pulsatile LH secretion. Blood samples were allowed to clot overnight at 4 C, and serum was harvested and stored at -20 C until assayed. Anestrous studies were performed between April and July, and breeding season work was conducted between November and January. All procedures involving animals were approved by the West Virginia University animal care and use committee.
Experimental design
Exp 1. In this experiment we used tissue collected during
anestrus for an earlier study that examined the effects of
E2 on Fos expression in DA neurons (12). Briefly,
ewes were ovariectomized, and 3 weeks later, 0.5 cm long blank (n
= 4) or E2-containing SILASTIC brand implants
(n = 5; Dow Corning Corp., Midland, MI) were inserted
sc. One week later, frequent blood samples were collected for 6 h,
and the ewes were then given two iv injections of 25,000 IU heparin, 10
min apart. The animals were killed with an overdose of pentobarbital,
and their heads were rapidly removed and perfused with 6 liters 4%
paraformaldehyde in 0.1 M phosphate buffer containing 10 IU
heparin/ml and 0.1% NaNO3. Brains were removed,
and the appropriate tissue block was dissected out, postfixed for
24 h, and then infiltrated with 30% sucrose in phosphate buffer.
Frozen coronal section of the hypothalami were cut at 60 µm and
stored in cryopreservative at -20 C. Seven to nine selected sections
per ewe were immunostained using a dual fluorescence procedure for
ER and Fos/FRA. The sections were selected based on gross anatomical
landmarks to examine ER-containing neurons in the following areas
(18): lateral septum, organum vasculosum laminae terminalis (OVLT),
preoptic area (POA), anterior hypothalamic area (AHA), ventromedial
hypothalamus, and arcuate nucleus (ARC). ER-containing cells were
labeled using rat monoclonal antibodies against human ER (H222,
Abbott Laboratories, North Chicago, IL), and Fos was
identified with rabbit polyclonal antibodies directed against amino
acids 128–152 of Fos (K-25, Santa Cruz Biotechnology, Inc., Santa Cruz, CA). These antibodies recognize both Fos and
FosB as well as the Fos-related antigens, FRA1 and FRA2.
Exp 2. This experiment determined whether there was a
seasonal variation in the ability of E2 to induce
Fos in a subset of ER-containing neurons identified in Exp 1.
Animals were ovariectomized during the breeding season (n = 10) or
anestrus (n = 9), and 0.5-cm long SILASTIC brand implants
containing E2 (n = 5 ewes/season) or blank
implants (n = 5 in breeding season, n = 4 in anestrus) were
inserted sc 3 weeks later. Frequent blood samples were collected for
4 h at 1 week after implant insertion. After the completion of
blood collection, ewes were heparinized and killed with an overdose of
pentobarbital, their heads were perfused, and tissue was collected and
processed as in Exp 1.
In this experiment we only analyzed a subset of ER-containing
neurons in the medial POA (mPOA), at the level of the OVLT. The
following anatomical landmarks were used to identify the appropriate
section for each ewe: 1) bilateral anterior commissures, 2) the optic
chiasm, 3) the supraoptic recess of the third ventricle, and 4) the
OVLT. In this experiment ER was identified using mouse monoclonal
ER antibodies (DAKO Corp., Carpenteria, CA), that give
a stronger signal than the H222 antibodies in ovine tissue (15). These
monoclonal antibodies were produced against human ER using
splenocytes of BALB/c mice immunized with recombinant ER (19).
Dual fluorescence immunostaining procedure
After three 5-min washes in phosphate buffer containing 0.1%
Triton-X (PBTX), tissues were placed into 0.1 M glycine (in
PBTX) for 30 min to remove excess aldehydes. After a set of three 5-min
washes in PBTX, sections were placed into 5% goat blocking serum
(Jackson ImmunoResearch Laboratories, Inc.) in PBTX for
1 h and then coincubated overnight at 4 C on a shaker table with
rabbit Fos antibodies (1:5000) and rat ER antibodies (1:50 for Exp
1) or mouse ER antibodies (1:200 for Exp 2) in PBTX containing 5%
normal goat serum. After an overnight wash in PBTX at 4 C on a shaker
table, the Fos antibodies were conjugated to goat antirabbit
IgG-fluorescein isothiocyanate (FITC), and ER antibodies were
conjugated to either goat antirat IgG-tetramethyl rhodamine
isothiocyanate (TRITC; Exp 1) or goat antimouse IgG-TRITC (Exp 2)
during a 30-min incubation at room temperature (all second antibodies
diluted 1:50). After three 5-min PBTX washes, tissues were coincubated
with rat IgG (Exp 1) or mouse IgG (Exp 2) and rabbit IgG (10 µg/ml
each) to form a bridge to which additional fluorescently labeled
antibodies could be attached. Blocking serum, second antibodies, and
IgGs were obtained from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA). The incubations with fluorescent
secondary antibodies and IgGs were repeated three times to form
multiple bridges and increase the intensity of the fluorescent
staining. Tissues from both treatment groups in Exp 1 and all four
treatment groups in Exp 2, were processed simultaneously. Both the H222
(18) and the DAKO Corp. (15) ER antibodies and the
anti-Fos serum (12) have been validated for use in ovine neural tissue;
we confirmed that controls that omitted one of the primary antibodies
completely eliminated the appropriate fluorescence without obviously
affecting the intensity of the other fluorescent probe. Although the
cross-reactivity of the two ER antibodies with ERß has not been
directly tested, the differences in the regional distribution of ERß
messenger RNA (17) and ER antigen (15, 18) imply that these
antibodies do not detect ERß.
Analyses
Tissue analysis. Sections were mounted onto gelatin-coated
microscope slides and coverslipped using Fluoromount mounting
medium (Southern Biotechnology Associates, Birmingham, AL). Images of
immunostained sections were acquired using a Carl Zeiss
microscope (New York, NY) and image analysis software from Biological
Detection Systems (Pittsburgh, PA). Individual Fos- and ER-labeled
cells were counted in each analyzed region by an investigator unaware
of the particular treatment or the animal identification number.
Counting criteria, based on the size and shape of nuclear labeling,
were cross-checked between the two investigators responsible for
counting cells. Dual labeled cells were identified using the BDS
software to overlay the two images and produce a spectral combination
of green (fluorescein) and red (rhodamine) that resulted in
yellow-marked dual labeled cells. ER and Fos colabeling was then
confirmed using side by side images of the individual ER and Fos
micrographs and visually identifying cells that contained both the
ER label and the Fos label with respect to microscopic tissue
landmarks (Fig. 1
). Results were
expressed as the percentage of ER-positive cells that also contained
Fos.
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Statistical analysis. The effects of
E2 treatment on LH pulse frequencies, within
season, were determined by the Wilcoxon-Mann-Whitney test. In Exp 1,
percentages of ER-positive cells that contained Fos, within an
anatomical region were compared between treatments, using the
t test. The effects of E2 treatment on
total numbers of Fos-positive and ER-immunoreactive cells in each
region, were also analyzed by t test. In Exp. 2, the
influence of E2 and season on the number of
Fos-positive cells, the number of ER-positive cells, and the
percentage of ER-positive cells containing Fos were statistically
evaluated by two-way ANOVA. Statistical significance was set at
P < 0.05 for all analyses.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-containing neurons expressing Fos (17.8% vs. 1.7%)
in the mPOA (Fig. 2). Estradiol had no
significant effect on colocalization of ER and Fos in any of the
other hypothalamic areas (Fig. 2) analyzed, including the more caudal
POA (sections that included the decussation of the anterior
commissure). The increase in Fos expression in ER-containing
neurons was limited largely to a group of neurons in the ventromedial
portion of the mPOA (Fig. 3). These cells
were near the OVLT, but clearly not in this structure.
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-containing neurons and of those
expressing Fos in all of the analyzed regions are presented in Table 1. Treatment with
E2 did not alter the number of cells expressing
ER in any area, and the only significant change in Fos expression
was a decrease in the number of cells immunostained for this antigen in
the lateral septum of the E2-treated ewes.
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-containing
neurons in the mPOA at the level of the OVLT (Fig. 4). Levels of Fos expression in these
ER-positive cells were similar between seasons in untreated
ovariectomized ewes. However, E2 treatment
produced a 3-fold increase in Fos expression in these cells during
anestrus, whereas it had no effect during the breeding season. In fact,
there was a slight decrease in the percentage of ER-containing cells
expressing Fos in E2-treated ewes during the
breeding season, but this change was not statistically significant.
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). There were approximately 25% more
ER-positive cells in this area during anestrus than in the breeding
season, but this was not a statistically significant difference (Fig. 5). Treatment with E2 did not alter the number of
ER-positive cells in either season.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-containing cells are found in a number of regions throughout the
ovine hypothalamus (18, 23). Despite this wide distribution, only a
subset of these cells, in the mPOA at the level of the OVLT, reacted
with significant Fos activation after 7 days of
E2 treatment of anestrous ewes. This selective
activation of ER-positive cells may reflect in part the low dose of
E2 used in these experiments, which does not
induce a LH surge or estrous behavior (24); some of the other
ER-containing areas of the hypothalamus have been implicated in
these actions of E2 (25, 26). Data from Exp 2
revealed that activation of these ER-positive cells in the mPOA is
seasonal in nature; only during anestrus do these cells respond to this
physiological E2 treatment with a significant
increase in the percentage that express Fos.
The higher counts of ER-positive and Fos-positive cells in Exp 1
than in Exp 2 was somewhat surprising, but two factors may have
contributed to this difference. First, a more selected area was
analyzed in Exp 2 than Exp 1, so that not all of the ER- and
Fos-positive cells in the mPOA were counted. Second, different sources
of primary antibodies against ER and different lots of secondary
antibodies were used, and tissues from Exp 1 and Exp 2 were processed
in different laboratories (University of Cincinnati and West Virginia
University, respectively). Despite these technical differences and the
different antibodies to ER used in these two experiments, the
stimulatory effects of E2 on Fos expression in
this subset of ER-containing cells were remarkably similar.
One potential problem in interpreting these data are that Fos is
generally thought to reflect acute neural activation (27), whereas
these experiments quantified Fos after 7 days of
E2 treatment. This treatment was chosen because
it activated A15 DA neurons in previous work (12) and because more
acute treatments, with higher doses of E2, would
also stimulate other neural systems, such as those involved in
triggering the preovulatory GnRH surge (28). Nevertheless, the chronic
treatment does create a possible discrepancy between the timeline and
the expected physiological window of Fos activation. However, there is
some evidence that Fos-related antigens, which are also detected by the
antibodies used in these studies, can be useful markers of prolonged
neuronal activation (29). It is possible that acute
E2 treatment might have activated other
ER-containing regions or that E2 might
stimulate ER-containing neurons without increasing Fos or FRA
expression. Thus, these data do not preclude a role for other neural
systems. Nevertheless, they do demonstrate that ER-containing
neurons in the mPOA clearly respond to physiological concentrations of
E2 that inhibit LH secretion during anestrus.
The correlation between activation of these ER-containing neurons
and inhibition of LH pulse frequency by E2 raises
the possibility that these neurons participate in
E2 negative feedback of GnRH secretion during
anestrus. If these neurons do participate, they may project directly to
GnRH neurons. In the ewe, neurons near the OVLT project to the more
dorsal portions of the mPOA (30) that contain many GnRH perikarya. In
the rat, neurons from this region innervate GnRH neurons (31), but at
this time there is no direct evidence for similar connections in the
ewe. Alternatively, these neurons could act indirectly, by stimulating
the A15 DA neurons that are known to participate in
E2 negative feedback during anestrus. Preliminary
findings that ER-containing neurons in this region project to the
A15 region (32, 33) support this alternative. It is important to note,
however, that the postulated role for these ER-containing neurons in
E2 negative feedback is not consistent with the
report that local E2 implants in the mPOA did not
inhibit LH pulse frequency in anestrous ewes (34). However, these
implants were dorsal to the ER-containing neurons identified in this
study, and the volume of tissue affected by such
E2 implants is unknown. To further complicate
this situation, recent work reported that similar
E2 implants into the mPOA do inhibit LH pulse
frequency (26). However, this study was performed in breeding season
ewes, so its relevance to E2 actions in anestrous
ewes is unclear.
There is also evidence that E2 acts in the A15
area; local E2 implants into this region
decreased LH pulse frequency in ovariectomized ewes during anestrus
(34). However, the inhibitory effects of these implants were relatively
modest compared with those of systemic E2
treatment (34), suggesting that this steroid may act at other areas in
the brain to inhibit GnRH pulse frequency. Nevertheless, these data
raise the possibility that E2 acts directly upon
A15 DA cells that do not appear to contain ER, just as it stimulates
DA synthesis in the striatum, a region that does not contain detectable
ER (35). This effect could be due to nongenomic actions of this
steroid, as a membrane-associated ER has been described (36, 37).
However, the time course of the negative feedback actions of
E2 in anestrus (12, 38) is more consistent with a
nuclear action. Moreover, the membrane ER of the rat is recognized by
one of the monoclonal antibodies (H222) (37) used to identify the
nuclear ER in sheep (14) (Exp 1). Thus, it is more likely that any
direct action of E2 on A15 DA cells is via ERß
(16). This receptor has a distribution different from that of ER in
the rat hypothalamus (39, 40), and the messenger RNA for ERß is
present in the retrochiasmatic area of the sheep (17). However, recent
data suggest that the ERß protein is not present in A15 DA neurons of
the ewe (41).
One possible explanation for the seasonal variation in
E2-induced Fos expression comes from the
observation that there are approximately 20% more cells in the POA
expressing ER in anestrous, than in breeding season, ewes (15). The
same study demonstrated the absence of season-dependent changes in
numbers of ER-positive neurons in the VMN or ARC. These data raise
the possibility that a discrete cell group in the mPOA may be induced
to express ER during inhibitory photoperiods, so that they are
selectively stimulated by this steroid in anestrus. It is interesting
to note that we observed a similar 25% increase in ER-containing
neurons in the mPOA during anestrus, although this change failed to
reach statistical significance.
In conclusion, we have demonstrated that ER-containing cells in the
mPOA, at the level of the OVLT, respond to E2
with an increase in activity, as measured by Fos, during anestrus, but
not in the breeding season. These changes correlate with seasonal
changes in activity of A15 DA neurons, suggesting that these two areas
may be coupled. However, the role of these ER-containing neurons in
seasonal breeding remains to be elucidated.
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Acknowledgments |
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Footnotes |
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2 Present address: Babcock GSM, Wake Forest University, Winston-Salem,
North Carolina 27109.
3 Present address: Department of Biomedical and Health Sciences,
Grand Valley State University, Allendale, Michigan 49401.
Received July 20, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Adrenergic neurons inhibit luteinizing hormone pulse amplitude in
breeding season ewes. Biol Reprod 54:380–386[Abstract]
containing afferents to the hypothalamic A15 region in the
ewe. Soc Neurosci Abstr 25:1452 (Abstract)
and -ß mRNA in the
rat central nervous system. J Comp Biol 388:507–525
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