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) Distribution in Hamster and Sheep Brain: Colocalization in Gonadotropin-Releasing Hormone and Other Identified Neurons1
Department of Cell Biology, Neurobiology, and Anatomy, University of Cincinnati College of Medicine (H.T.J., M.N.L.), Cincinnati, Ohio 45267; the Department of Biology, University of Massachusetts (L.S.L.), Amherst, Massachusetts 01003; Istituto di Endocrinologia, Università di Pisa (E.M.), Pisa, Italy; and the Department of Medicine, University of Chicago Medical Center (L.J.D.), Chicago, Illinois 60637
Address all correspondence and requests for reprints to: Dr. Heiko T. Jansen, Department of Cell Biology, Neurobiology, and Anatomy, University of Cincinnati College of Medicine, P.O. Box 670521, Cincinnati, Ohio 45267-0521. E-mail: jansenht{at}e-mail.uc.edu
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Abstract |
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THR) colocalization in two seasonal breeders, the golden
hamster and the sheep. THR was identified in hamster and sheep brain
by Western blot analysis. Furthermore, THR immunoreactivity was
widely distributed in brain and was colocalized in identified
populations: GnRH neurons (hamster, 28%; sheep, 46%); dopaminergic
neurons of the A14 (hypothalamic) and A16 (olfactory bulb) cell groups,
but not in the hypothalamic A13 cell group; and
neurophysin-immunoreactive neurons of the supraoptic and
paraventricular nuclei. The finding of THR in GnRH and A14 dopamine
neurons provides an anatomical substrate for direct thyroid hormone
action on the reproductive neuroendocrine system of these two
seasonally breeding species. It remains to be determined whether the
GnRH gene itself or the gene of another constituent within the same
GnRH neuron is responsive to thyroid hormones. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It is well established that the developing nervous system is extremely
sensitive to thyroid hormones. The active hormone,
L-T3, acts on the nervous system and other
organs by binding to specific receptors encoded by the protooncogene,
c-erbA (10, 11). These thyroid hormone receptors (THRs)
belong to the superclass of nuclear hormone receptors (12) and can
alter gene transcription in both the presence and absence of ligand
(T3) (see Refs. 13 and 14 for reviews). Two THRs exist (
and ß), and these are further subdivided into alternatively spliced
products to yield 1-, 2-,
ß1-, and ß2-isotypes. The distribution of
THR protein and messenger RNA (mRNA) in brain has been documented
(15, 16, 17, 18, 19, 20, 21, 22, 23), but relatively little information exists as to the specific
phenotype of THR-containing neuronal populations (18, 20, 23),
especially those potentially involved in reproductive neuroendocrine
function. Indeed, as indicated above, it is not clear whether the
effects of thyroid hormones on the reproductive axis reflect central or
peripheral actions. Evidence to support a central site of thyroid
hormone action derives from studies demonstrating that thyroid hormone
placed directly into the brain decreases TRH mRNA (24) and secretion
(25), suppresses pituitary TSH release (26), and affects short
day-induced gonadal regression in hypothyroid male hamsters (27).
Furthermore, the effects of thyroidectomy on the reproductive axis are
mediated by the GnRH system (9, 28) and can be reversed by
T4 replacement (29). These observations strongly implicate
the neuroendocrine axis as a target site of thyroid hormones.
Two recent observations raise the possibility that T3 acts
directly on GnRH neurons. First, GnRH/LH release increases after
thyroidectomy (28); this can be completely reversed by peripheral
T4 administration (29). Second, GnRH gene expression in the
frog (Xenopus laevis) increases at the time of
metamorphosis, coincident with the surge in thyroid hormones and THRs
(19, 30, 31). Taken together, these findings suggest that the GnRH
system may be a potential target of thyroid hormone action. Compelling
evidence for a direct effect of thyroid hormones on the GnRH neuron
would, at a minimum, require colocalization of THRs within GnRH
neurons. Thus, the primary objective of this study was to determine
whether GnRH neurons contained THRs. To this end, GnRH neurons in two
seasonal breeders, the male golden hamster and the female sheep (both
sensitive to thyroid hormone manipulations) (4, 7, 27), were examined
for THR colocalization using dual label immunocytochemistry (ICC). As
the -isoform is the most abundant THR expressed in brain, its
overall distribution in brain was examined in addition to
colocalization within identified neuronal populations. The results
confirm widespread THR immunoreactivity in both hamster and sheep
brain and reveal colocalization within GnRH, dopaminergic, and
vasopressin/oxytocinergic neurons.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Western analysis
THR was identified in hamster and sheep forebrain by SDS-PAGE
using a Bio-Rad Protean II dual slab apparatus. A 12% running gel and
a 4% stacking gel were prepared, and the gels were loaded with 15–35
µg protein. Proteins were resolved at 50 mA for approximately 60 min
and then transferred to nitrocellulose membranes. Proteins on gels were
visualized using Coomassie blue staining. Membranes were blocked
overnight at 4 C with 3% BSA in PB containing 0.9% sodium chloride
(PBS) and on the following day were transferred to primary antiserum
(-144, diluted 1:500) in PBS containing 3% BSA for 24 h at 4
C. Membranes were then washed in PBS, four times for 5 min each time,
followed by a 1-h incubation in horseradish peroxidase (HRP)-conjugated
donkey antirabbit IgG (Jackson ImmunoResearch Laboratories, West Grove,
PA; diluted 1:50,000) in PBS. After additional washes in PBS (four
times for 5 min each time), the antibody complexes were visualized
using the Vector VIP substrate kit (Vector Laboratories, Burlingame,
CA) according to the manufacturer’s instructions.
ICC
Free floating sections were processed for single and dual
label ICC using a modified avidin-biotin-HRP procedure (Vectastain
Elite kit, Vector Laboratories) as described previously (33). Briefly,
sections were washed with PB containing 0.1% Triton-X 100 (Sigma
Chemical Co., St. Louis, MO) and then incubated for 10 min in 1%
hydrogen peroxide to quench endogenous peroxidase activity. After a 1-h
incubation in 0.1 M glycine and washes, sections were
incubated in 1% blocking serum (goat) for 1 h and then placed
directly into primary antibody (THR) containing 4% blocking serum
and 2% BSA. Sections were incubated in primary antibody overnight at 4
C with constant gentle agitation. On the following day, sections were
washed and then incubated in biotinylated goat antirabbit IgG (1:200;
Jackson ImmunoResearch) for 1 h. For THR, the avidin-HRP
complex was visualized with nickel-enhanced diaminobenzidine as the
chromagen to yield a blue-black reaction product. In double label
preparations, THR was visualized as described above, and then the
sections were washed overnight in PB containing 0.1% Triton-X 100
followed by another overnight incubation in additional antibody against
either GnRH, neurophysin (NP), or tyrosine hydroxylase (TH). The second
antigen was visualized using unenhanced diaminobenzidine to produce a
brown reaction product.
Antibodies
Anti-THR is a polyclonal antibody (-144) raised in rabbits
against a synthetic peptide corresponding to the D domain shared by
both 1- and 2THR isotypes and previously
shown to produce specific labeling of rat neurons (34); it was diluted
1:60,000. Anti-GnRH is a polyclonal antibody raised in rabbits (LR-1;
provided by Dr. Robert Benoit) and was diluted 1:50,000. Anti-NP is a
rabbit polyclonal (Incstar, Stillwater, MN) and was diluted 1:5000.
Anti-TH is a rabbit polyclonal (EugeneTech International, Ridgefield
Park, NJ) and was diluted 1:2000.
The specificity of the THR antiserum (-144) was initially
described using in vitro expressed THR and in rat brain
sections (34). In the present study Western blot analysis was used to
confirm the ability of -144 antiserum to detect THR protein in
sheep and hamster brain (see above). Antibody specificity was further
evaluated by incubating hamster and sheep sections in antiserum
preadsorbed with synthetic peptide (10–100 µg; Affinity BioReagents,
Neshanic Station, NJ). The specificities of GnRH, NP, and TH antisera
in sheep and hamster have been described previously (35, 36, 37, 38).
Analysis
The regional distribution of THR immunoreactivity was
evaluated and scored according to the relative densities of
immunopositive nuclei. Dopaminergic neurons (TH-immunoreactive) of the
A13, A14 (hypothalamus), and A16 (olfactory bulb), but not of other
dopamine cells groups, and vasopressin/oxytocinergic
(NP-immunoreactive) neurons of the hypothalamic paraventricular (PVN)
and supraoptic nuclei were examined for colocalization; quantitation
was not performed. Quantitative comparison of GnRH and THR
colocalization was performed in every fourth (hamster) and every sixth
(sheep) section and included the medial preoptic area and hypothalamus,
regions previously shown to contain the greatest number of
GnRH-immunoreactive neurons in both species (35, 39). Approximately 100
GnRH neurons from each of 3 sheep and 3 hamsters were examined for
colocalization; the location of each single and double labeled GnRH
neuron was recorded on standardized brain drawings.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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THR as determined using Western analysis (Fig. 1
). Both 48- and 58-kDa bands (-1 and
-2 THR, respectively) were visualized and thus confirm the broad
specificity of this antibody. The 58-kDa band was always more intensely
stained in both species and most likely reflects the greater affinity
of the antibody for 2THR than for 1THR
(34).
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THR immunoreactivity-144 antibody was confirmed by
preadsorption with synthetic peptide or omission of primary antisera.
Preadsorption eliminated virtually all nuclear staining in both species
(Fig. 2). As previously reported (34),
normal rabbit serum alone produced a small amount of nuclear staining
and, therefore, reflects nonspecific interactions. Nuclear THR
immunoreactivity was readily identified in many regions of the hamster
and sheep brain, and these results are summarized in Table 1. Within the olfactory bulb, dense
accumulations of THR immunoreactivity were found in granule cells,
mitral cells, and within clusters surrounding individual glomeruli.
Many labeled nuclei were observed within the frontal cortex (Fig. 2)
and in the preoptic area at the level of the organum vasculosum of the
lamina terminalis (Table 1). Nuclear THR staining was observed in
virtually all hypothalamic subdivisions to varying degrees (Table 1).
Especially dense labeling was observed within the arcuate and
ventromedial hypothalamic nuclei. Within the suprachiasmatic nucleus
staining was moderate and exhibited significant intranuclear variation
in staining intensity (Fig. 3). Dense
nuclear labeling was also seen in the anterior pituitary of both
species (not shown). In cerebellum, the granule cell layer of both
species contained the greatest numbers of immunoreactive nuclei, with
far fewer positive nuclei in the molecular layer (Fig. 4). Most sheep Purkinje cells contained
immunopositive nuclei (Fig. 4), an observation only rarely made in
hamster cerebellum. However, when present, the intensity of the
immunopositive hamster Purkinje cell nuclei was similar to that in the
sheep. In both species, prominent cytoplasmic staining of Purkinje
cells was evident (Fig. 4).
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THR immunoreactivity (Fig. 5 and Table 2). Analysis of GnRH neurons for
colocalization in 3 hamsters (346 GnRH neurons total) and 3 sheep (378
GnRH neurons total) yielded double labeling in 28% and 46% of GnRH
neurons, respectively. There was no apparent regional difference in the
percentage of double labeled GnRH neurons in either species (Table 2).
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THR immunoreactivity in both hamster
and sheep (Table 2
). Nearly all A16 neurons of both species contained
immunopositive nuclei (not shown). In both species many hypothalamic
A14, but none of the hypothalamic A13 neurons, contained
THR-positive nuclei (Table 2 and Fig. 5).
NP.
NP-immunoreactive neurons within both the supraoptic
nuclei and PVN of both species contained THR-immunopositive nuclei
(Table 2). Similar to observations made for the dopaminergic neurons,
not all NP-immunopositive neurons contained THR-positive nuclei.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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THR in the brain (15, 16, 19). Because
the antibody we used recognizes both 1 and
2THRs (34) (by Western blot in the present study), we
cannot attribute the immunostaining solely to a particular receptor
isotype. However, the distribution of THR immunoreactivity we
observed is very similar to that previously described in the rat by
Bradley et al. (15) using 2THR-specific
probes. Together, the previous results and those from the present study
of high levels of THR in olfactory bulb, forebrain, and hypothalamus
suggest that the majority of -receptors identified in the sheep and
hamster brain were of the 2-subtype. This proposition is
further supported by the Western blot results and the earlier finding
that only approximately 25% of 1THR protein can be
immunoprecipitated with the -144 antibody (34). Furthermore, the
recent cloning of sheep THRs and subsequent Northern analysis confirmed
the higher levels of 2THR transcripts relative to
1 transcripts in sheep brain (40). To date, similar
studies have not been performed in hamsters. The widespread
distribution of THR immunoreactivity in both hamster and sheep brain
suggests potentially complex interactions between THRs and many
different neuronal populations.
The physiological significance of these high levels of THR expression
in the adult brain remains controversial. It is generally believed that
the adult brain is much less sensitive to thyroid hormones than the
newborn, and it has been proposed that the high levels of
2THR (nonhormone binding) may be responsible for this
reduced sensitivity (41). If, in fact, this is the case, and the
neuropeptide- and neurotransmitter-containing neurons we identified
contained 2THR, then presumably these cells would be
unresponsive to thyroid hormones. However, this interpretation must be
viewed with caution, as the DNA-binding ability of the
2THR isotype affords it the unique opportunity to bind
to thyroid hormone response elements (TREs) of genes independently of
ligand to influence gene expression (14, 41, 42, 43). Regardless of this
possibility, apparently not all cells within a population of identified
neurons contained THR. Thus, it remains a distinct possibility that
these cells express other THR isotypes capable of binding thyroid
hormone and altering gene transcription. In support of this possibility
we have gathered preliminary evidence from the sheep indicating that
the ß2THR isotype is also widely distributed in brain and
colocalized within identified neurons such as those containing GnRH
(Jansen, H. T., unpublished).
The finding of THR colocalization in GnRH neurons raises the intriguing possibility of a direct influence of thyroid hormone on GnRH gene expression, as has been recently suggested to occur in Xenopus (30). The onset of GnRH gene expression in Xenopus corresponds temporally with the metamorphic peak of thyroid hormones (30) and the time of peak THR gene expression (31). The precise interaction between thyroid hormones and GnRH gene expression in mammalian species remains to be clarified, and identification of a TRE within the GnRH gene as well as other genes coexpressed within the GnRH neuron is required to put the current observations into perspective. It should also be noted that the colocalization in sheep and hamster GnRH neurons does not necessarily indicate a role in seasonal reproduction per se. Although the species used in the present study exhibit seasonal reproductive cycles that are strongly influenced by thyroid hormones, the GnRH neurons of other species may also contain THRs; this awaits verification.
The colocalization of THR in TH-immunoreactive neurons (A14 and A16) confirms earlier findings made by Puymirat et al. (23) in cultured dopamine neurons. Tyrosine hydroxylase activity is up-regulated in hypothyroid animals (44), and dopaminergic neurons may, therefore, represent targets of thyroid hormones. With respect to seasonal reproductive activity, the hypothalamic A14 dopamine cell group has been shown to play an important role in seasonal estradiol negative feedback responses (45, 46). For example, in the ewe, A14 neurons are activated in response to estradiol during anestrus but not during the breeding season (45), and destruction of these neurons results in an attenuated response of the reproductive neuroendocrine axis to estradiol during the nonbreeding season (46). The similarity between these lesion effects and those seen after thyroidectomy in the ewe (4) suggests that thyroid hormones may interact with the dopaminergic system to modify seasonal changes in sensitivity to estradiol-negative feedback.
Colocalization of THR in NP neurons expands the list of identified
neuronal populations containing THRs in other species (15, 18, 20).
Recently, it has been shown that oxytocin, but not vasopressin, gene
expression is directly regulated by T3 and THR
in vitro and in vivo (47). These findings lend
additional support to the idea that thyroid hormones play an important
role in modulating neuroendocrine function.
Thyroid hormone responses involve interactions between THRs and an appropriate nucleotide motif (TRE) within the 5'-regulatory region of genes (14). Although the number of genes known to contain TREs continues to grow, three candidates are particularly relevant to our discussion of the reproductive neuroendocrine axis and thus are especially worthy of consideration. First, as described above, the GnRH gene may be directly affected by thyroid hormones. Second, the gene for the peptide galanin in rodents is coexpressed in many GnRH neurons (48), undergoes dynamic fluctuation throughout the estrous cycle (49), and is expressed during GnRH neuron development and migration (50). In addition, hypothyroidism reduces galanin concentrations in the median eminence and mRNA levels in the PVN (51, 52). Third, dopaminergic neurons (i.e. expressing the TH gene) are important for seasonal reproductive transitions and undergo morphological and biochemical alterations in response to thyroid hormones in vitro (23). Unfortunately, until TREs are identified within the upstream regulatory regions of these genes, any direct effect of thyroid hormones on galanin and TH expression remains inconclusive. However, unlike galanin and TH, the promoter regions of rat and human GnRH genes contain motifs resembling estrogen receptor/THR response elements (53, 54). Furthermore, the rat GnRH promoter also contains a retinoic acid response element (54); retinoic acid response elements can interact with THRs alone or with THR/retinoic acid receptor heterodimers (14). Additional studies are now required to fully elucidate the interactions between THRs and responsive elements within the GnRH gene as well as potential interactions between THRs and other genes coexpressed in the GnRH neuron. Such experiments may reveal novel and potentially important mechanisms, either direct or indirect, for regulating the physiology of the GnRH neuron.
A potentially significant observation made in sheep, but less apparent
in the hamster, was the frequent apposition of small (4- to 5-µm)
THR-immunoreactive nuclei and GnRH somas. Given their size, these
small nuclei most likely represent glial cells. The importance of these
close appositions is highlighted by the recent finding that synaptic
input onto sheep GnRH neurons varies with season, and these changes
seem to be associated with alterations in glial ensheathment (55).
Whether this plasticity in synaptic input/glial ensheathment in sheep
is mediated by thyroid hormones remains to be determined. Glia contain
THRs (17, 22) and clearly play important roles in the plasticity of the
adult nervous system while also being strategically positioned to
modulate neuroendocrine function (56). Thus, it is possible that
seasonally breeding species whose reproductive transitions are
dependent upon thyroid hormones may share a common mechanism of
neural/glial plasticity associated with changes in reproductive
neuroendocrine status.
In summary, the present study confirms widespread THR distribution
within the hamster and sheep brain. By colocalizing THR in GnRH and
A14 dopamine neurons, the substrate exists for a direct action of
thyroid hormone on the reproductive neuroendocrine system of these
seasonal breeders. Further studies are needed to demonstrate that
thyroid hormones delivered directly to the brain can mimic their
peripheral effects on seasonal reproduction.
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Acknowledgments |
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Footnotes |
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Received April 4, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and ß thyroid hormone receptor genes
in rat brain and pituitary. Proc Natl Acad Sci USA 86:7250–7254 and
ß mRNAs in regions of adult rat brain. Mol Cell Endocrinol 70:13–20[CrossRef][Medline]
and ß
ovine thyroid hormone receptor cDNAs. Thyroid 6:237–243[Medline]
-related protein which
binds DNA acid but does not bind thyroid hormone. Mol Endocrinol 2:893–901[Abstract]
isoforms generated by alternative splicing differentially activate
myosin HC gene transcription. Nature 334:539–542[CrossRef][Medline]
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