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Department of Physiology (F.J.K.), Reproductive Sciences Program (C.V., H.J.B., F.J.K.), University of Michigan, Ann Arbor, Michigan 48109-0404; Department of Cell Biology, Neurobiology and Anatomy, University of Cincinnati College of Medicine (H.T.J., L.C., M.N.L.), Cincinnati, Ohio 45267-0521; Department of Biological Sciences, Kent State University (J.D.G.), Kent, Ohio 44240-0001; and Division of Pediatric Cardiology, Department of Pediatrics, Rainbow Babies and Childrens Hospital, Case Western Reserve University (M.W.), Cleveland, Ohio 44106-6011
Address all correspondence and request for reprints to: Dr. F. J. Karsch, Reproductive Science Program, University of Michigan, 300 North Ingalls Building, Room 1101 SW, Ann Arbor, Michigan 48109-0404. E-mail: fjkarsch{at}umich.edu
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Abstract |
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Introduction |
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Despite extensive evidence for neuroplasticity within the adult GnRH system, the underlying molecular basis remains unknown. In the neurohypophyseal-magnocellular system, proteins that influence neuronal remodeling, in particular the neural cell adhesion molecule (NCAM), are implicated in plasticity during periods of altered neurosecretion (14, 15, 16). Of interest, NCAM and its polysialylated isoform (PSA-NCAM), which promotes plasticity by modifying the stability of cell-cell contacts, are expressed along the prenatal migratory path of GnRH neurons (17, 18). The importance of these molecules to the migratory process is suggested by findings that GnRH neuronal migration is perturbed by immunoneutralization of NCAM or by enzymatic alteration of its PSA isoform (18, 19, 20). Both NCAM and PSA-NCAM continue to be expressed in the adult brain and, of particular interest to the present study, are found in regions that contain GnRH perikarya, fibers, and terminals (21, 22, 23). Further, the expression of NCAM and PSA-NCAM in the preoptic area and hypothalamus of Siberian hamsters is influenced by photoperiodic manipulations that alter GnRH secretion (24). All of these findings are consistent with a role for NCAM and PSA-NCAM in remodeling of the GnRH system. Nevertheless, it has not been assessed whether these molecules exist within or immediately adjacent to GnRH neurons in the adult brain.
In this study, we explored the potential for PSA-NCAM-mediated plasticity in the GnRH neurosecretory system of the adult ewe, a model in which seasonal changes in both structure and function of the GnRH system are well documented (25). As a first step, we examined whether PSA-NCAM is expressed within or directly adjacent to GnRH neurons. Having found that it is, we determined whether PSA-NCAM expression associated with GnRH neurons varies in conjunction with seasonal alterations in GnRH neuronal function.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Monitoring reproductive neuroendocrine state
All ewes were ovariectomized at least 4 months before death. Two
weeks before collection of tissues, a 3-cm sc estradiol implant
(26) was placed into each ewe to restore a luteal phase
concentration of circulating estradiol (2–3 pg/ml) (27).
Blood was sampled daily by jugular venipuncture from the first day of
estradiol replacement until death to monitor LH secretion as an index
of seasonal reproductive state. During anestrus, the GnRH
neurosecretory system is exquisitely sensitive to estradiol negative
feedback and thus pulsatile GnRH and LH secretion are dramatically
suppressed within a few days after placing the estradiol implants
(27, 28). In contrast, animals in the breeding season are
much less responsive to estradiol negative feedback, and GnRH and LH
secretion remain high.
Blood samples were centrifuged to remove cells, and serum was stored at -20 C. LH was assayed in duplicate 20- to 200-µl aliquots using a modification (29) of a previously reported RIA (30, 31). Concentrations are expressed in terms of NIH LH-S12. The sensitivity for 200 µl (95% confidence interval of buffer control) was 0.58 ng/ml. All samples were run in a single assay; mean intraassay coefficients of variation for serum pools displacing radiolabeled LH to approximately 30% and 65% of buffer control values were 6.7% and 14.2%, respectively.
Immunocytochemistry
Sodium heparin (25,000 U, iv) was injected at 10 min and 1 min
before euthanasia with a barbiturate overdose (Beuthanasia,
Schering-Plough Animal Health Corp., Kenilworth, NJ). Ewes
were immediately decapitated, both carotid arteries were catheterized,
and the head was perfused with 4 liters 2% paraformaldehyde in 0.1
M sodium phosphate buffer (pH 7.4) containing 10,000
U/liter heparin and 0.01% sodium nitrite. After perfusion, the brain
was removed, and a block containing the whole hypothalamus and preoptic
area (from 5 mm rostral to the optic chiasm to the anterior pole of the
mammillary bodies) was isolated and postfixed overnight at 4 C in the
same fixative. The block was washed in PBS (pH 7.3) and immersed in
10% sucrose for 24 h at 4 C and then in 20% sucrose containing
0.01% sodium azide at 4 C until sectioning. Serial 50-µm coronal
sections were obtained using a freezing microtome. Sections were stored
at -20 C in a cryopreservative (32).
Every fifth section was stained for GnRH and PSA-NCAM immunoreactivity. Sections were washed in 0.01 M PBS and then incubated for 30 min at room temperature in 0.1 M glycine in PBS and 0.2% Triton X-100. After washing, sections were preincubated for 45 min at room temperature in a blocking solution (PBS, 2% BSA, 3% normal goat serum, 0.2% Triton X-100, and 0.01% sodium azide). Sections were exposed to primary antiserum to GnRH (LR1, provided by Dr. Robert Benoit, McGill University, Montréal, Québec, Canada) at a dilution of 1:10,000 and antibody to PSA-NCAM (5A5, monoclonal mouse IgM directed against the 2–8-linked polysialic acid of NCAM, provided by Dr. Urs Rutishauser, Sloan–Kettering Institute, New York) at a dilution of 1:250 in the blocking solution. Sections were incubated for 72–96 h at 4 C. After washing, sections were incubated for 90–120 min in a mixture of Alexa 594-conjugated goat antirabbit IgG (Molecular Probes, Inc., Eugene, OR) at a dilution of 1:500 and goat antimouse fluorescein isothiocyanate-conjugated IgM µ-specific (Sigma, St. Louis, MO) at a dilution of 1:250. Sections were washed, mounted, and coverslipped using Prolong (Molecular Probes, Inc.) mounting medium for fluorescence. Stained sections were examined with a x60 oil immersion lens on a confocal microscope (MRC-600, Bio-Rad Laboratories, Inc., Hercules, CA). For each GnRH neuron, a series of serial optical sections (z-series, 1- to 1.5-µm thickness) was generated. In addition, a series of GnRH confocal images was generated from the median eminence of one ewe in each group.
In addition to aforementioned tissue processing, several additional
sections through the median eminence from these ewes were processed
using an avidin-biotin immunofluorescent procedure involving tyramide
amplification (33). Specifically, sections were washed in
0.1 M PBS, blocked with 1%
H2O2 in 0.1 M
PBS for 10 min, and washed again with PBS. This was followed by
incubation in a blocking solution of 0.1 M PBS containing
0.1% Triton X-100 and 4% normal donkey serum for 1 h. Sections
were then incubated overnight (
18 h) at room temperature with a
cocktail of primary antibodies to PSA-NCAM (5A5 diluted 1:250 in the
blocking solution) and GnRH (LR-1 diluted 1:3000). After this, sections
were washed in PBS and incubated with biotin-conjugated donkey
antimouse IgG (Jackson ImmunoResearch Laboratories, Inc.,
West Grove, PA) diluted 1:200 in the blocking solution for 1 h.
The sections were again washed in PBS, incubated with an avidin-biotin
complex (ABC-elite kit, Vector Laboratories, Inc.,
Burlingame, CA) diluted 1:200 in PBS for 1 h, washed again in PBS,
and incubated in biotin-conjugated tyramide (NEN Life Science Products, Boston, MA) diluted 1:250 in PBS and 0.003%
H2O2 for 10 min. This was
followed by another wash, incubation in CY2-conjugated streptavidin
(Jackson ImmunoResearch Laboratories, Inc.) diluted 1:100
in PBS for 30 min, another wash with PBS, and incubation for 30 min
with CY3-conjugated donkey antirabbit IgG (Jackson ImmunoResearch Laboratories, Inc.) diluted 1:100 in PBS. Sections were then
washed in 0.1 M phosphate buffer, mounted, and
coverslipped. Images were obtained using a laser scanning confocal
microscope (LSM 510, Carl Zeiss, New York, NY) at x10 and
x63 magnification. Overall, findings using the initial procedure were
confirmed, including the elimination of PSA-NCAM immunoreactivity by
omission of the primary antibody or treatment of tissue sections with
endoneuraminidase enzyme (see below). The modified method, however,
provided better definition of the PSA-NCAM immunoreactive signal. This
procedure was used for the results presented in Fig. 4
, which
illustrates PSA-NCAM/GnRH distribution in the median eminence.
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Data analysis
Confocal images from the z-series of each individual GnRH neuron
were analyzed using NIH Image software. Both image channels (GnRH and
PSA-NCAM) could be viewed simultaneously and thus analyzed at the same
time. The extent of PSA-NCAM immunoreactivity associated with GnRH
neurons was quantified using two separate methods by different
investigators working independently of each other and without knowledge
of the experimental group. For both methods, analysis of GnRH neurons
was performed using three consecutive z-sections in which a nuclear
profile was present.
The first analysis determined the percentage of the perimeter of the GnRH cell body juxtaposed to PSA-NCAM immunoreactivity (cell body perimeter excluded interfaces of dendrites/soma and axon/soma). Fifteen to 32 GnRH somas in the preoptic area were analyzed for each ewe. Two parameters were compared between breeding and anestrous seasons using unpaired t test: 1) mean percentage of the perimeter of GnRH somas juxtaposed to PSA-NCAM immunoreactivity, and 2) percentage of GnRH somas contacted at least 90% by PSA-NCAM immunoreactivity.
The second analysis was based on the estimated density of PSA-NCAM immunoreactivity associated with GnRH neurons. For each z-section, a 150 x 150-pixel circle was first centered over a GnRH neuron, and a threshold value based on the average pixel intensity for the entire GnRH channel was applied. Then, the number of pixels (pixel area) occupied by each GnRH neuron was calculated for each z-section. An area of the exact same size was analyzed in the corresponding PSA-NCAM channel with threshold values based on the average pixel density for that channel, and an estimate of the density of PSA-NCAM immunoreactivity was calculated as the number of pixels. This value was also used to calculate a ratio of PSA-NCAM density to GnRH somal area. In addition, an estimate of PSA-NCAM density was calculated in an area of the optical field devoid of GnRH immunoreactivity. For each ewe, mean values derived from the three consecutive z-sections examined were determined for 1) the ratio of PSA-NCAM density to GnRH somal area; 2) the density of PSA-NCAM immunoreactivity associated with GnRH neurons; and 3) the density of PSA-NCAM immunoreactivity not associated with GnRH neurons. Statistical comparisons between the two seasons were made using an unpaired t test.
For both types of analysis, parameters that do not change with season and/or reproductive neuroendocrine state were also measured as an assessment of analysis consistency: 1) GnRH cell nuclear diameter for the first type of analysis; and 2) GnRH somal area (expressed as pixels) for the second method.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). In
contrast, LH did not decrease after estradiol treatment in breeding
season animals.
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, A vs. B, lower
panel shows example for median eminence; similar results were
obtained in other areas of the hypothalamus). GnRH immunostaining was
not eliminated by endo-N treatment (Fig. 2, A and B, upper
panels), although the number of samples examined was insufficient
to assess whether GnRH immunoreactivity was quantitatively altered.
Omission of either primary antibody in the presence of both secondary
antibodies eliminated all staining corresponding to the omitted primary
antibody (not illustrated).
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, A–D). Occasional GnRH neurons
did not exhibit associated PSA-NCAM immunoreactivity even though they
were located in the vicinity of GnRH neurons that did (Fig. 3B).
PSA-NCAM immunoreactivity was associated primarily with the periphery
of the GnRH cell soma and the large proximal and distal dendrites and
less frequently around axons and small diameter dendrites (Fig. 3C).
When GnRH neurons were juxtaposed, the interface between them, whether
somato- somatic, dendro-somatic, or axo-somatic, was invariably
PSA-NCAM immunoreactive (Fig. 3D). PSA-NCAM-positive structures that
appeared as rings of immunoreactivity but did not exhibit GnRH
immunoreactivity (e.g. asterisk in Fig. 3A) were
seen in proximity to some GnRH neurons (31% of GnRH neurons analyzed
were juxtaposed to such structures). Whether this pattern of PSA-NCAM
labeling represented immunoreactivity associated with non-GnRH neurons
or glial cells was not clear.
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, A and C), associated with a region of
intense GnRH-immunoreactive axons/terminals (Fig. 4, B and C). This
region also contained fibers in which PSA-NCAM and GnRH
immunoreactivity overlapped (Fig. 4D, long arrows) as well
as PSA-NCAM-immunoreactive fibers not associated with GnRH labeling
(Fig. 4D, large arrowheads) and GnRH immunoreactivity not
associated with PSA-NCAM labeling (Fig. 4D, small
arrowheads).
Seasonal comparison
According to the first method of analysis, the percentage of the
perimeter of GnRH somas juxtaposed to PSA-NCAM immunoreactivity
(i.e. percentage of PSA contact) was significantly greater
in the breeding season than in anestrus (P < 0.02),
although the absolute difference was relatively small (Fig. 5A). Similarly, the percentage of GnRH
somas contacted at least 90% by PSA-NCAM immunoreactivity was greater
in the breeding season than in anestrus (42.7 ± 5.0% of GnRH
neurons in the breeding season vs. 16.5 ± 5.3 in
anestrus; P < 0.02; Fig. 5B). Seasonal differences
were confirmed by the second analysis. The ratio of PSA-NCAM to GnRH
labeling and the density of PSA-NCAM immunoreactivity associated with
GnRH neurons were 23% and 38% greater (P < 0.05),
respectively, in the breeding season than in anestrus (Fig. 5, C and
D). However, the density of PSA-NCAM immunoreactivity in POA areas
devoid of GnRH immunostaining did not exhibit any seasonal differences
(Fig. 5E). Neither GnRH soma size nor nuclear diameter exhibited a
seasonal difference (data not illustrated).
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Discussion |
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Several aspects of the morphological relationship between PSA-NCAM immunoreactivity and GnRH neurons are noteworthy. The first is the extent of this relationship; nearly all GnRH neurons examined in the preoptic area were associated with PSA-NCAM immunoreactivity. Second, PSA-NCAM immunoreactivity was observed at the periphery of GnRH perikarya, consistent with NCAM being a cell surface protein that is polysialylated on its extracellular domain (38). However, an ultrastructural study will be required to determine whether PSA-NCAM is contained within the plasma membrane of GnRH neurons or within other cells or neural elements in apposition to GnRH neurons. If PSA-NCAM is, in fact, expressed on the extracellular surface of GnRH perikarya, this might explain why GnRH neurons are relatively less innervated than adjacent nonidentified neurons (13, 39), given evidence to suggest that PSA-NCAM impedes the formation and stabilization of synapses (40). Our finding that PSA-NCAM is rarely associated with small diameter GnRH dendrites complements earlier observations that these processes are the major sites of synaptic inputs to ovine GnRH neurons (39).
The present and previous observations are consistent with the possibility that PSA-NCAM is functionally significant to seasonal plasticity of the GnRH neurosecretory system of adults. In the Siberian hamster, photoperiodic manipulations that cause seasonal shifts in gonadotropin secretion also alter the relative amounts of PSA and other NCAM isoforms in the basal hypothalamus, a region important to GnRH regulation (24). Our present findings in sheep extend those observations by demonstrating that the expression of PSA-NCAM is intimately associated with preoptic GnRH neurons and that this expression varies with the seasonal shift in reproductive neuroendocrine activity. Specifically, we observed a significant difference in three indexes of the PSA-NCAM/GnRH relationship: the extent to which GnRH neurons are juxtaposed to PSA-NCAM immunoreactivity, the ratio of PSA-NCAM to GnRH immunoreactivity, and the density of PSA-NCAM immunoreactivity associated with GnRH neurons. Each of these measures was greater during the breeding season than during anestrus. Further, this seasonal difference appeared to be at least partially specific to GnRH neurons, because PSA-NCAM immunoreactivity in adjacent preoptic areas devoid of GnRH neurons and fibers did not change with the season. Although our findings are consistent with the possibility that PSA-NCAM may contribute to the seasonality of the GnRH system, it should be noted that in themselves they do not demonstrate that this molecule plays a role in seasonal changes in either the structure or function of GnRH neurons. Such a conclusion requires evidence that seasonal alterations in the GnRH system can be prevented by perturbation of endogenous PSA-NCAM expression by endoneuraminidase or other means.
It is important to note that our study was conducted in ovariectomized ewes treated with constant release estradiol implants. Thus, the seasonal difference in PSA-NCAM expression associated with GnRH neurons was not the consequence of an altered ovarian steroid milieu, which in itself can induce hypothalamic neuroplasticity (5, 6, 7, 8, 10). Rather, it reflects an intrinsic seasonality in expression of the molecule. Prior work in this animal model has demonstrated that in the face of an unvarying gonadal steroid milieu, there are marked seasonal changes in both synaptic inputs to GnRH neurons and the pulsatile secretion of GnRH into pituitary portal blood (13, 28). The seasonal difference in GnRH secretion was confirmed indirectly in the present study by serum LH concentrations; values were not reduced by estradiol in the breeding season, but they were profoundly suppressed during anestrus.
Both the direction and the magnitude of the seasonal difference in PSA-NCAM associated with GnRH neurons are noteworthy. Regarding magnitude, this seasonal difference was relatively small. Although it could be argued that such a subtle difference is not likely to have significant functional consequences, it is important to note that a minute change in NCAM polysialylation can dramatically alter the physical and functional properties of the molecule (38, 41). Further, it should be noted that the tissues analyzed in the present study were obtained several months before or after the seasonal reproductive transition, not during the actual transition, when the shift in GnRH secretion occurs and the PSA-NCAM difference might be greater. In addition, it is important to emphasize that a seasonal change in PSA-NCAM itself might not be critical. Rather, its mere presence in or around GnRH neurons may promote neuroplastic remodeling, as suggested for the neurohypophyseal magnocellular system, where its expression is constitutive and its action permissive (15, 16).
With regard to direction of the seasonal change, we observed greater expression of PSA-NCAM associated with GnRH neurons in the breeding season than during anestrus. Our results in sheep are consistent with findings in Siberian hamsters that the expression of PSA-NCAM in hypothalamic regions that regulate reproduction is more prevalent under a stimulatory photoperiod than under an inhibitory day length (24). It is of interest that synaptic input to GnRH neurons is also greater during the breeding season than during anestrus (13). This raises questions related to the role that PSA-NCAM may play in promoting seasonal changes in the innervation of GnRH neurons.
Although it is recognized that the PSA isoform of NCAM promotes neuroplasticity by modifying the stability of cell to cell contacts, its specific role in synaptic rearrangements is not well understood. On the one hand, it has recently been suggested that PSA-NCAM can impede the formation of synapses or reduce their stability (40). This was based on observations that genetic deletion of PSA-NCAM or enzymatic removal of PSA from NCAM enhanced synaptic number in the pyramidal cell layer of the hippocampus. On the other hand, it has been suggested that PSA-NCAM can promote the formation of synapses onto neuroendocrine cells of the hypothalamic magnocellular system, where PSA-NCAM was found to be present in both neurons and adjacent glial cells (16). In this system, lactation and dehydration induce both the withdrawal of glial processes surrounding oxytocin and vasopressin neurons and an increase in synaptic input to these neurons; this is associated with enhanced hormone release (16). Enzymatic removal of PSA from NCAM blocked both the withdrawal of glial processes and the increased synaptic input. It was suggested, therefore, that PSA-NCAM promotes the innervation of magnocellular neurons by acting in either a permissive or a stimulatory fashion, possibly involving glial withdrawal (16). It is also important to note that the expression of PSA-NCAM is activity dependent (15, 16, 38). Therefore, the higher levels of PSA-NCAM immunoreactivity associated with GnRH neurons in the breeding season might be a consequence, rather than a cause, of the increased GnRH neurosecretory activity at this time of year.
In addition to influencing synaptic rearrangements, PSA-NCAM might affect the GnRH system via alternative mechanisms. For example, PSA-NCAM might promote rearrangements of cells in the proximity of GnRH neurons, cells that could influence neurosecretion by paracrine mechanisms. Paracrine regulation of a reproductive neuroendocrine transition has been implicated at the time of puberty in rats; in this case, growth factors secreted by astrocytes appear to be critically important for activating GnRH neurons (42, 43). The present study did reveal extensive PSA immunostaining in structures located near GnRH neurons and fibers, but which themselves were not immunoreactive for GnRH. Further research is required to identify these structures and their possible relationship to the secretory activity of GnRH neurons.
In summary, our findings demonstrate that a close anatomical association exists between PSA-NCAM and GnRH neurons in the adult ewe. This holds both for the median eminence, where GnRH fibers and terminals are juxtaposed to PSA-NCAM immunoreactivity, and the preoptic area, where GnRH perikarya are extensively contacted by PSA-NCAM. Further, the expression of PSA-NCAM associated with preoptic GnRH neurons changes seasonally, being greater during the period of enhanced GnRH secretion in the breeding season than during anestrus. These findings provide evidence that PSA-NCAM is well positioned anatomically to promote remodeling within the GnRH axis, and they encourage future work to establish the functional significance of this molecule to both the structure and the secretory activity of the GnRH system in adults.
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Acknowledgments |
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Footnotes |
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2 Present address: INSERM, U-501, IFR Jean Roche,
Faculté Nord, boulevard Pierre Dramard, 13916 Marseilles Cedex
20, France.
3 Present address: Department of Veterinary and Comparative Anatomy,
Pharmacology and Physiology, Washington State University College of
Veterinary Medicine, P.O. Box 646520, Pullman, Washington
99164-6520.
Received July 7, 2000.
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expression to discrete loci of the neuroendocrine
brain induces female sexual precocity. Proc Natl Acad Sci USA 94:2735–2740This article has been cited by other articles:
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). Ewes were killed to collect
brains for immunocytochemical analysis after the last blood sample.
) or anestrus (AN,
). Data on the left are the results of the first data
analysis and depict the percentage of the perimeters of GnRH somas
exhibiting PSA-NCAM immunoreactivity, i.e. PSA contact
(A), and the percentage of GnRH somas that are contacted at least 90%
by PSA-NCAM immunoreactivity (B). Data on the right are
the results of the second method of analysis and depict the ratio of
PSA-NCAM density to GnRH somal area (C), the density of PSA-NCAM
immunoreactivity associated with GnRH perikarya (D), and the density of
PSA-NCAM immunoreactivity not directly associated with GnRH perikarya
(E). For each type of data analysis, measurements were performed on
three consecutive sections of a z-series for each neuron. Results are
presented as the mean ± SEM (n = 4 ewes for both
breeding and anestrous seasons). *, P 
0.02 for
the first method of data analysis and P