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Department of Clinical Veterinary Science, University of Bristol, Langford House, Langford, BS40 5DU, United Kingdom; and Institut National de la Recherche Agronomique, Physiologie de la Reproduction des Mammiferes Domestiques, Nouzilly 37380, France
Address all correspondence and requests for reprints to: Donal Skinner, Department of Clinical Veterinary Science, University of Bristol, Langford House, Langford, BS40 5DU, United Kingdom. E-mail: donal.c.skinner{at}bristol.ac.uk
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Abstract |
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Introduction |
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The ovine model has provided a wealth of detailed information on the hypothalamic release of GnRH (7), and studies investigating the effects of progesterone on GnRH release have revealed both acute (6) and chronic (8) effects of progesterone exposure. Ovine GnRH perikarya are located in regions (9) shown previously to bind progesterone (10), but research on other species suggests that most GnRH neurons do not express steroid receptors (11, 12, 13, 14). It is hypothesized, therefore, that the effects of progesterone are transduced to the GnRH perikarya through an interneuronal system. Visualization of the neural location of the ovine PR is a fundamental step toward understanding the regulation of GnRH secretion and uncovering the identity of neurotransmitter systems involved in its control. However, it has proved difficult (Lehman, M. N., University of Cincinnati, Cincinnati, OH, and A. E. Herbison, The Babraham Institute, UK; personal communication), despite success in several other species (15, 16, 17, 18).
The difficulty in visualizing the neural ovine PR could be due to an impotence of available antibodies to recognize, in the sheep, the epitopes to which they have been raised or that the ovine PR is fundamentally different to that found in other species. Alternatively, methodological artifacts such as the fixation of the tissue may lie at the heart of the problem. In several studies that have visualized PRs in other species, tissue was fixed using acrolein (19, 20). Moreover, several studies have already noted that neural steroid receptors appear to be acutely fixation sensitive (19, 21, 22). These two suggestions may not be mutually exclusive; if the ovine PR was recognized weakly by an antibody and was also fixation sensitive, then it may be undetectable using available antibodies and standard fixation protocols.
The first objective of this study, therefore, was to attempt to visualize the PR in the ovine brain following different fixation protocols. It was recently reported that GnRH neurons may express steroid receptors (23) and, thus, the second objective of this study was to carry out colocalization investigations between the PR and GnRH.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Does fixation affect visualization of the PR?
Brains were fixed with 3 liters of either 4% paraformaldehyde
(n = 4) or 4% acrolein (n = 4) in PBS (pH 7.4, 0.1
M), infused using a pump over a period of 20 min in a
ventilated fume hood. A block containing the preoptic area (POA) and
the hypothalamus was dissected out and placed in the same fixative for
either 2 h (n = 2 per fixative) or 24 h (n = 2 per
fixative) before immersion in 30% sucrose PBS (pH 7.4). Six identical
sets of 60 µm-thick coronal sections (i.e. each section
within a set was 360 µm apart from the preceding section) were cut on
a freezing microtome from the septum through to the caudal hypothalamus
at the level of the mammillary bodies. Sections were stored in
cryoprotectant (24) at -20 C until immunocytochemistry
was performed.
Single-labeling immunocytochemistry
Free-floating sections were washed (3 x 5 min) in 0.05
M Tris-buffered saline (TBS, pH 7.8), transferred to 40%
methanol/1% H2O2/TBS
solution (10 min), washed, and transferred to 20% normal goat
serum/0.1% Triton/TBS for 1 h. Sections were then incubated in
one of two different monoclonal mouse antihuman PR antibodies (1:20 -
1:100; 72 h): PgR Ab-8 (Neomarker, Union City, CA) or NCL-PGR
(Novocastra, Newcastle, UK). At room temperature, sections were washed,
incubated in secondary antiserum (1:300; 90 min; biotinylated goat
antimouse IgG; Vector Laboratories, Inc., Burlingame, CA),
washed and placed in Vectastain Elite kit (1:50; 90 min;
Vector). Visualization of immunoreactivity was performed with
nickel-DAB as described (11). Sections were mounted on
gelatinized slides and coverslipped.
Can PRs be retrieved using antigen-unmasking techniques?
Using the PgR Ab-8 antibody, acrolein fixation allowed
visualization of the neural ovine PR, but colocalization studies could
not be performed as background staining was exceptionally high. Using a
modification of an approach pioneered by Shi et al.
(25), we went on to ask whether PRs in
paraformaldehyde-fixed tissue could be unmasked using high temperature
antigen retrieval (HTAR). Free-floating sections were boiled (15 min)
in HTAR solution (pH 6.8; 100 C; Vector Laboratories, Ltd., Petersborough, UK) and left to cool in the unmasking
solution (20 min). The PR (PgR Ab-8 1:1000) was then visualized
using the described procedure. Boiling tissue in either water or TBS
was ineffective at unmasking the PR. We did not investigate whether the
unmasked PR was detectable by the NCL-PGR antibody.
For mapping the distribution of the ovine PR, brains from four additional ewes were perfused with paraformaldehyde, followed by 24 h postfixation and processed as described above.
Do GnRH neurons express PRs?
A set of sections from each of four ewes was stained for PR and
used for double-labeling immunocytochemistry by washing in 40%
methanol/TBS/1% H2O2
solution for 10 min and then placing in polyclonal rabbit anti-GnRH
(1:40 000; LR1, gift of R. Benoit, Montréal, Canada) for 48
h. At room temperature, sections were placed in secondary antibody
(1:300; 90 min; biotinylated goat antirabbit IgG; Vector Laboratories, Ltd.), washed and placed in
Vectastain Elite kit (1:50; 90 min; Vector Laboratories, Ltd.). Immunoreactivity was detected using DAB
(11).
Several GnRH neurons appeared to express PRs using this approach. However, it was recognized that if the PR immunoreactive cells are faintly stained and immediately above the GnRH perikarya, then it is difficult to discriminate between the DAB and the Ni-DAB using conventional light microscopy. To resolve this dilemma, an additional set of PR labeled sections were placed in anti-GnRH (1:5000) as before but then placed in FITC-labeled goat antirabbit IgG for 90 min.
Antibodies and controls
The monoclonal mouse antibody, PgR Ab-8, used in this study is a
mixture of two specific and well-characterized clones
(26): hPRa2 recognizes both the A and B forms of the human
PR and hPRa3 only the B form. PR from a human endometrial carcinoma
(EnCa 101) was used to generate the antibodies. These monoclonal
antibodies cross-react with the uterine PRs of the cow, mouse, and
rabbit (27). Cross-reactivity with the ovine PR has not
been established but inference from the partially sequenced ovine PR
suggests 96%, 94%, and 97% identical amino acid sequences with the
human, mouse and rabbit PRs, respectively (28).
Liquid-phase adsorption control experiments were performed by overnight incubation of PR antibody (1:3000) with recombinant human PR-A and PR-B (each at 0.05 mg/ml; D. Edwards, Denver, CO). In contrast to unadsorbed antiserum, no specific staining was detectable in ovine POA sections to which the adsorbed antibody was applied. Omission of primary antisera and substitution with an inappropriate secondary antiserum also abolished specific staining.
The specificity of the LR1 antiserum has been established previously in the sheep (12). Comparison of immunoreactive product intensity and the number of detectable neurons on adjacent POA sections that were either unmasked or untreated, demonstrated that antigen unmasking did not affect the detection of GnRH-immunoreactive (-IR) perikarya with this antiserum.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Can PRs be retrieved using antigen-unmasking techniques?
Using HTAR, the PR became clearly visible in
paraformaldehyde-fixed hypothalamic sections, which had been postfixed
in paraformaldehyde for either 2 h or 24 h. Even at a
dilution of 1:3000 the clarity of PR-IR cells was superior to those in
acrolein fixed tissue. HTAR did not improve immunodetection of PR in
acrolein-fixed tissue.
Figure 1
shows the distribution of PR-IR
cells in a representative ewe brain. Cells immunoreactive for the PR
were identified throughout the POA extending from the region of the
organum vasculosum of the laminae terminalis to the rostral arcuate
nucleus. PR-IR cells were located predominantly in medial and central
aspects of the POA adjacent to the third ventricle (Figs. 1A and 2A)
and extended into the anterior hypothalamic area (Figs. 1B and 3, A and
B) with a few cells in the paraventricular nucleus (Fig. 1, C and D,
2B). Few cells were detected in the bed nucleus of the stria terminalis
(Fig. 1B), and none were found in the supraoptic nucleus. In one ewe, 4
PR-IR cells were located in the ventrolateral septum; none were found
in other ewes. Within the ventrolateral-ventromedial hypothalamus, a
continuum of PR-IR cells extended from the region medial to the fornix
(Figs. 1, E and F, and 2D) toward the arcuate nucleus. Most PR-IR cells
were then confined to the arcuate nucleus (Figs. 1G and 2C). Very few
PR-IR cells were detectable in the caudal arcuate nucleus in the region
of the mammillary recess (Fig. 1H and 2, E and F). A range of lightly
to intensely labeled PR-IR was observed in each region, with
immunoreactivity restricted to the nucleus and the nucleolus was
unstained.
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) and thus mounting the
sections is more cumbersome.
Do GnRH neurons express PRs?
In the ventral region of the POA, GnRH neurons were found within a
sea of PR-IR cells (Fig. 3A). More
dorsally and laterally within the section, there were less PR-IR cells,
although several GnRH-IR neurons were still evident (Fig. 3B). The
majority was clearly not progesterone receptive (Fig. 3, B, C, and E).
The initial results using Ni-DAB and DAB to visualize the PR- and
GnRH-IR cells, respectively, suggested that a few GnRH neurons express
PRs (Fig. 3D). If a PR-IR nucleus is positioned immediately above a
GnRH perikaryon (Fig. 3E), and also faintly stained (see nuclei
surrounding GnRH perikaryon on Fig. 3D), then the contrast between the
DAB of the GnRH and the Ni-DAB of the PR is poor, making it difficult
to state unequivocally that the neuron expresses PR. This problem is
eliminated when using fluorescence to detect GnRH perikarya (Fig. 3, F
and G) and may explain why, when using FITC for detecting GnRH neurons,
only a single perikaryon of 732 identified GnRH perikarya was
positively colocalized with the PR.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The distribution of PR-IR cells in the ovine brain agrees with an earlier investigation where progesterone binding was identified in the POA and mediobasal hypothalamus (10). The PR distribution reported in the present study also concurs largely with a recent study on ovariectomized estrogen-treated ewes (30) but, in contrast to our study, a significant number of PR-expressing cells were also detected in the supraoptic nucleus and in the caudal arcuate nucleus, below the mamillary recess. This variance may be due to a difference in affinity of the antibodies for the ovine PR or may reflect the use of different steroid treatments, as the ewes in our study had been exposed to progesterone for 10 days before killing.
The distribution of PR-containing cells in the POA-hypothalamic continuum of sheep shows a high degree of similarity with the PR distribution reported in the rat (31), mouse (20), guinea pig (15), primate (16), mink (17), and cat (18). In all species studied, the presence of intense immunoreactivity in the POA, ventrolateral zone of the ventromedial nucleus, and arcuate nucleus is a consistent feature. These sites are known to be critically important in the steroid-mediated regulation of gonadotropin secretion and reproductive behavior.
While the distribution of PR-IR cells in the ovine brain described in our study overlaps with that of the estrogen receptor (ER), PR-IR cells appear more restricted in their distribution: no IR cells were found in the supraoptic nucleus and almost none in the ventrolateral septum. This more restricted distribution of the PR, compared with the ER, has also been noted in the guinea pig (15), mink (17), and cat (18). The number of PR-IR cells detected in the ovine hypothalamus in the present study appears to be substantially less than that reported for the ER (cf, 11, 12). In the guinea pig, PR-IR nuclei have only been observed in ER-IR cells, and not all ER-IR cells express the PR (32). This may explain the apparent quantitative difference between the PR and ER in the ewe but confirmation of this hypothesis will require PR-ER colocalization studies under the same steroidal conditions.
This study provides evidence that progesterone does not target GnRH
perikarya directly to inhibit GnRH secretion. The near absence of
GnRH-PR colocalized neurons in our study agrees with reports on the
monkey (16) and mink (17) and supports the
hypothesis that GnRH perikarya are not directly affected by steroids.
Nevertheless, it is possible that more GnRH neurons, than the single
perikaryon we observed, express PRs. In this respect, the hypothesis
that GnRH neurons do not express ERs has been challenged by a report
showing some rat GnRH neurons expressing ERs (23) and,
more importantly, a study using single cell RT-PCR on mouse GnRH
neurons, which demonstrated the presence of both ERß and ER
transcripts (14). If this finding is extrapolated to the
PR, then it is plausible to speculate that low levels of functional PRs
may be expressed by mammalian GnRH neurons but that these PRs may be
undetectable using immunocytochemical techniques. Furthermore, it is
possible that the antibody used in the present study, which recognizes
both the A and B forms of the human PR, only detected the ovine PR-B
and, thus, any GnRH neurons expressing PR-A would have remained
undetected. It is also worth noting that a small, but centrally
positioned, population of GnRH-PR neurons has been reported in the
guinea pig (19) and, as only few GnRH neurons may be
involved in pulse generation (33), it is plausible to
speculate that inhibition of only a few GnRH neurons may be sufficient
to inhibit GnRH secretion.
However, if our immunocytochemical data showing the near absence of GnRH-PR colocalization are taken together with in vivo studies on the ewe, then our data favor the hypothesis that GnRH neurons are not directly targeted by progesterone. First, this study was conducted on tissue taken from ewes at a time when progesterone rapidly and specifically inhibits GnRH secretion through the PR (6). Second, when progesterone was administered directly into ventromedial hypothalamic region, either through microimplantation (34) or microinfusion (35), it inhibited LH secretion, whereas administration into the POA was ineffective. Thus, progesterone is more likely to act via an interneuronal system to inhibit GnRH secretion but its identity remains to be elucidated: candidates include the opioidergic, GABAergic, and catecholaminergic systems (36, 37).
Several researchers have noted that both the neural (21, 22) and nonneural (38) ER are fixation sensitive.
Lehman et al. (see Fig. 1 in Ref. 21) showed clearly that
the intensity of ER staining and, thus, the number of detectable
cells, was substantially higher when hypothalamic tissue was
immediately placed in sucrose than when tissue was first postfixed in
paraformaldehyde for 24 h before placement in sucrose. It is also
noteworthy that McEwen and Alves (22) reported that the
type of fixative was a factor affecting visualization of the ERß.
Preliminary investigations (Skinner, D. C., and M. Goubillon,
unpublished data) indicate that HTAR increases the number of positively
identified ER immunoreactive cells in ovine hypothalamic tissue that
had been postfixed in paraformaldehyde for 24 h. It is noteworthy
that the detection or improved visualization of neural steroid
receptors by HTAR may not be confined to the steroid receptors since
McQuaid et al. (39) noted that the revelation
and intensity of several antigens in the human CNS were markedly
improved by microwave treatment. On the other hand, the ability of an
antibody to detect its target antigen (e.g. GnRH, present
study) may be unaffected by antigen retrieval. It is also possible that
the high temperature may damage or destroy certain antigens. It will be
essential, therefore, to conduct comparative studies between
HTAR-treated and -untreated tissue for every antibody.
It is not known how antigen retrieval works, which is not surprising because it is still not known how paraformaldehyde fixes tissue and, therefore, masks tissue antigenicity (25). Shi et al. (25) proposed that antigen masking may result from a change in protein conformation. Because antibodies recognize specific epitopes localized in a particular spatial configuration within a protein molecule, a change in this spatial configuration may render the antigen invisible to the antibody. HTAR may restore this spatial configuration.
In summary, using an antigen unmasking approach, this study describes the distribution of PR neurons in the ovine brain at a time when progesterone potently suppresses GnRH secretion. Importantly, it also provides strong evidence that this suppression must be indirect since GnRH perikarya do not express the PR.
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Acknowledgments |
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Footnotes |
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Received August 4, 2000.
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References |
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and ß messenger ribonucleic acids in adult
gonadotropin-releasing hormone neurons. Endocrinology 140:5195–5205 gene-disrupted mice. J
Neurosci 18:9556–9563-immunoreactivity in gonadotrophin-releasing
hormone-expressing neurones. J Neuroendocrinol 11:331–335[CrossRef][Medline]
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