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Endocrinology Vol. 138, No. 6 2585-2595
Copyright © 1997 by The Endocrine Society

ARTICLES

Effects of Photoperiod on Estrogen Receptor, Tyrosine Hydroxylase, Neuropeptide Y, and ß-Endorphin Immunoreactivity in the Ewe Hypothalamus

Donal C. Skinner1 and Allan E. Herbison2

Laboratory of Neuroendocrinology, The Babraham Institute, Cambridge CB2 4AT, United Kingdom

Address all correspondence and requests for reprints to: Dr. Allan E. Herbison, Laboratory of Neuroendocrinology, The Babraham Institute, Cambridge, CB2 4AT, United Kingdom. E-mail: allan.herbison{at}bbsrc.ac.uk


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The neural components underlying the influence of photoperiod upon reproductive functioning are poorly understood. In this study, we have used immunocytochemistry to examine whether changes in photoperiod may influence specific neuronal cell populations implicated in mediating gonadal steroid feedback actions on GnRH neurons. Short day (SD) exposed ewes in the midluteal stage of the estrous cycle and long day (LD) anestrous ewes were perfused in pairs and hypothalamic brain sections immunostained for tyrosine hydroxylase (TH), neuropeptide Y (NPY), ß-endorphin (ßE), and the estrogen receptor (ER). The number of ER-immunoreactive cells detected within the preoptic area, but not the hypothalamus, was approximately 20% higher (P < 0.05) in LD ewes compared with SD animals. The numbers of TH-immunoreactive neurons comprising the A12, A14, and A15 cell groups were not different between LD and SD ewes, and the percentage of A12 (~15%) and A14 (~25%) neurons expressing ERs was similarly unaffected by photoperiod. The number of ßE neurons detected in the arcuate nucleus was 50% lower (P < 0.05) in SD vs. LD ewes, whereas NPY-immunoreactive cell numbers in the median eminence were 300% higher (P < 0.05). Approximately 3% of NPY neurons in the median eminence, and 10% in the arcuate nucleus, expressed ER immunoreactivity in a photoperiod-independent manner. These studies indicate that changes in photoperiod may regulate ER expression within the preoptic area and suggest that hypothalamic NPY and ßE neurons are involved in the seasonal regulation of reproductive activity in the ewe.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE NEURAL mechanisms through which photoperiod influences the reproductive activity of seasonally breeding animals are not clear. In the sheep, it is established that part of the influence of changing day length on LH secretion profile occurs through alterations in gonadal steroid feedback mechanisms on the gonadotropic axis (1, 2). In particular, estrogen’s role in regulating LH secretion has attracted considerable attention and photoperiod-induced changes in estrogen’s feedback actions on GnRH neurons are thought to be critical in determining the onset and cessation of estrous cyclicity in the ewe (3). The efficacy of estrogen’s inhibitory feedback influence upon LH release varies substantially over the course of the year, whereas it acts principally to bring about the GnRH surge in the breeding season (3, 4). It seems likely, therefore, that the elucidation of the neural pathways used by estrogen to influence GnRH neurons in different seasons would provide considerable insight into both the photoperiodic regulation of reproduction and the gonadal steroid regulation of LH secretion.

The GnRH neurons do not express nuclear estrogen receptors (ERs) in either ovariectomized (5, 6) or anestrous (7) ewes and, at present, the only ER-expressing neuronal cell populations implicated in the seasonal regulation of ovine GnRH secretion are the hypothalamic dopaminergic and ß-endorphin (ßE) neurons and the GABA-containing cells of the preoptic area (POA) (5, 6, 7, 8, 9, 10, 11, 12, 13, 14). Although hypothalamic neuropeptide Y (NPY) neurons have only, as yet, been demonstrated to express ERs in the rat (15), a preliminary report has shown changes in NPY messenger RNA (mRNA) expression with season in the arcuate nucleus of the sheep (16). It is possible, therefore, that photoperiod may influence the biosynthetic and/or electrical activity of one or several of these neuronal cell populations to bring about the different seasonal effects of estrogen on GnRH neurons. We have begun to address this hypothesis by examining whether photoperiod influences the number of immunocytochemically detectable tyrosine hydroxylase (TH), ßE, and NPY cells in the hypothalamus of the ewe. As photoperiod may also influence ER expression within specific brain regions and neuronal cell populations, we have examined the effect of photoperiod on ER immunoreactivity within the hypothalamus and POA as well as within identified cell populations. In this study, we set out to provide a baseline set of data in the gonadal-intact animal and have performed experiments using paired, long day (LD)- and short day (SD)-exposed ewes. To ensure that levels of circulating estrogen were similar between the two groups, all SD animals were killed in the midluteal phase (11). Using this experimental model, we now report that substantial photoperiod-induced immunocytochemical changes occur within specific neurochemical- and ER-defined cell populations of the ovine hypothalamus and POA.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Twelve intact Clun Forest ewes from the Babraham Institute flock were maintained in artificial photoperiod rooms with free access to water and fed daily with hay, straw, and corn. Experiments were begun in the first and second weeks of January and performed on three groups of four ewes. All ewes were initially exposed to long days (LD: 16-h light, 8-h dark; lights on 04:00–20:00 h) for 90 days. Thereafter, half the ewes were maintained on long days and the other half were switched to short days (SD: 8-h light, 16-h dark; lights on 08:00–16:00 h) for 60–70 days. Reproductive status was determined by taking blood samples every 2 days for 3 weeks before perfusion and analyzing these samples for progesterone by RIA (Coat-A-Count; DPC, Los Angeles, CA). Ewes were killed in the morning in pairs (one LD and one SD) by an overdose of sodium pentobarbitone (Lethobarb, Duphar Veterinary, Southampton, UK), the head was severed, and catheters inserted into both carotid arteries. Approximately 0.5 liter of heparinized saline (75 IU/ml 0.9% NaCl) followed by 3 liters of 4% paraformaldehyde in PBS (pH 7.4) was infused into the carotids using a Masterflex pump (CP Instruments, Bishop’s Stortford, UK) over a period of 20 min. The brain was removed, and a block containing the POA and hypothalamus dissected and placed in the fixative solution for a further 2h before immersion in a 40% sucrose, Tris-buffered saline (TBS) solution (pH 7.4) for 1–2 days. Using a freezing microtome, six identical sets of 30 µm-thick coronal sections were cut from the rostral POA, beginning at the organum vasculosum of the lamina terminalis, through to the caudal hypothalamus at the level of the mammillary bodies.

Single-labeling immunocytochemistry
Free-floating sections were washed in a 40% methanol/TBS solution containing 1%H2O2 for 10 min before several TBS washes and incubation in either monoclonal mouse anti-ER (1:10; ID5, gift of G. Delsol, CHU Purpan, Toulouse, France), monoclonal mouse anti-TH (1:4 000, MAB 318, Chemicon, Harrow, UK), polyclonal rabbit anti-NPY (1:8 000, N-9528, Sigma, Poole, UK) or polyclonal rabbit anti-ßE (1:1 000, Affinity Research Products, Nottingham, UK) antibodies for 40–50 h at 4 C. Sections from the LD:SD paired animals were processed together in the same well. Three of the six sets of sections were incubated in either the TH, ßE, or NPY antibodies while two sets from each animal were placed with the ER antibody. After the primary antibody, sections were washed in TBS and incubated in secondary antisera (1:400 biotinylated horse antimouse for ER and TH, and 1:200 biotinylated goat antirabbit for NPY and ßE) for 90 min at room temperature. After further washes in TBS, the Vectastain Elite kit (1:50; Vector, Labs, Burlingame, CA, for ER) and peroxidase-labeled avidin-biotin complex (1:200; Amersham, Little Chalfont, UK for other antibodies) were applied for 90 min at room temperature. Visualization of immunoreactivity was performed with nickel-DAB as described previously (5). Each set of sections immunoreacted for TH, ßE, or NPY were mounted on gelatinized slides and coverslipped. Half of one set of sections immunoreacted for the ER were mounted.

Double-labeling immunocytochemistry
The remaining sections stained for the ER (one and a half sets per ewe) were divided into three equal lots and used for double-labeling immunocytochemistry by washing in 40% methanol/TBS/1% H2O2 solution for 10 min and placing in either the TH, NPY, or ßE antiserum for 40 h as described above. Secondary antibodies (1:400 peroxidase-labeled goat antirabbit for NPY and ßE and 1:400 peroxidase-labeled horse antimouse for TH; both from Vector) were applied for 2 h at room temperature and immunoreactivity detected using DAB immunocytochemistry without nickel (5). Sections were mounted and coverslipped.

Antibodies and controls
Production of the ER and TH antibodies has been reported (17, 18) and the latter used previously for identifying dopaminergic neurons in the sheep brain (19). The monoclonal mouse ID5 antibody is directed against the N-terminal region of recombinant human ER (17). Liquid-phase adsorption control experiments were performed by overnight incubation of the ID5 antibody (1:10; 4 C) with a 0.5 mg/ml concentration of an N-terminal mouse ER fragment (residues 1–182) generated by C. Chapman (The Babraham Institute, Cambridge, UK) from a GST-AF1 construct provided by M. Parker and F. L’Horset of Imperial Cancer Research Fund (London, UK). The specificity of the NPY antibody directed against porcine NPY has been established in the rat brain (20) and liquid-phase adsorption control experiments were performed in the present study by overnight incubation of 1 ml NPY antibody (1:8000; 4 C) with 10 nM of NPY (Sigma). The ßE antibody was raised against synthetic ßE and exhibits no cross-reactivity with enkephalin, substance P, vasoactive intestinal peptide, or calcitonin gene-related peptide. In all adsorption experiments, adsorbed and unadsorbed antibodies were applied to different sections from the same ewe. Other controls included the omission of primary antibodies from the incubation procedure in both single and double-labeling experiments. In all cases, the use of adsorbed antibodies or their omission resulted in a complete absence of specific immunostaining.

Analysis
The density of ER-immunoreactive nuclei within a brain region and the area of each stained nucleus were determined using a Leitz Laborlux S microscope coupled to a SeeScan Sonata 2 image analyzer (Seescan, Cambridge, UK). Sections from each LD:SD pair of ewes containing the rostral POA, ventrolateral division of the ventromedial nucleus (VMN), and arcuate nucleus (ARN) were matched, and all cells within a 0.5 mm by 0.5 mm square placed over the ER-immunoreactive cells were counted. The coronal levels at which the brain regions were analyzed are shown in Fig. 2Go. For each of the rostral POA, VMN and ARN, four brain sections containing the brain region of interest were selected at random from the half set of mounted ER-only stained sections and cells analyzed on both sides of the brain. As sections from the first pair of ewes were not immunoreacted with the ID5 antibody, a total of 5 SD and 5 LD ewes were evaluated. After subtraction of background gray levels, the image analyzer counted the total number of nuclear profiles within the field and then determined the area of staining for each object. Between 500 and 1500 nuclear profiles were analyzed for each brain region in each ewe.



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  		Figure 2. Low power photomicrographs of ER-immunoreactive cells in the (A) preoptic area, (B) ventrolateral division of the ventromedial nucleus, and (C) arcuate nucleus of a LD ewe. 3V, Third ventricle; F, fornix. Scale bar (A, B): 200 µm; (C) 100 µm.

 
The analysis of single TH, ßE, and NPY immunoreacted sections and double-labeled sections was undertaken by manual counting using a Leica DM RB microscope at 400-1000x objective power. The number of immunoreactive cell profiles in each population under investigation was counted in a minimum of four matched sections from each of the six LD:SD paired ewes. Analysis of the TH immunoreactivity was carried out at several levels through the POA and hypothalamus and referred to here using the nomenclature of Tillet and Thibault (21). The dopaminergic neurons of the A14 group were analyzed at two levels: the first at the level of the POA where positive cells are found within the organum vasculosum of the lamina terminalis (OVLT) and in the periventricular region and the second within the anterior hypothalamus where TH-immunoreactive cells are prominent in the ventral half of the periventricular area. These cells have a characteristic elongated bipolar morphology that is oriented parallel to the ventricle (21) and are clearly distinguishable from the A13 cells of the dorsomedial hypothalamus above and the A12 cells below. Cells immunoreactive for TH were also counted in the retrochiasmatic area of the anterior hypothalamus where they comprise the A15 cell group and in the rostral ARN where the A12 population predominates.

For the analysis of NPY and ßE immunoreactivity, sections containing the mediobasal hypothalamus were divided into those containing the rostral and caudal halves of ARN and immunoreactive cell numbers counted in a minimum of four sections from each half. As only one of the six sets of identical sections was reacted for each antibody, individual sections analyzed for NPY or ßE cell numbers were at least 150 µm apart in the coronal plane. Because many NPY-immunoreactive cell profiles were identified in the median eminence (ME), the number of positive cells in the caudal and rostral median eminence were also counted. In rostral sections, a line drawn along the edge of the ventricle and continued down through the ME was used to distinguish ARN (lateral to line) from ME (medial to line) cells. In caudal sections, the lateral boundaries of the ME are more readily apparent through the clear point of attachment that is observed between the ME and hypothalamus. Each cell exhibiting cytoplasmic immunoreactivity with a nuclear exclusion was considered a single cell profile.

The analysis of double-labeled sections was carried out in the same brain regions and manner as described above. In this case, the total number of TH- or NPY-immunoreactive cells with or without ER-immunoreactive nuclei was counted in a minimum of three brain sections from the five LD:SD pairs in each region of interest. In all cases, an average value was obtained for each ewe, and statistical analysis of paired LD:SD ewes (n = 5 or 6) was undertaken with Student’s paired t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Reproductive status
Plasma progesterone concentrations from an SD/LD pair of ewes are shown in Fig. 1Go. Progesterone concentrations above 1 ng/ml were indicative of the luteal phase and the time of the midluteal phase of the estrous cycle was calculated for each SD ewe. Anestrous ewes exhibited progesterone levels below 1 ng/ml for the whole 3-week period. Reproductive status was confirmed by visual inspection of the ovaries at autopsy.



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  		Figure 1. Representative progesterone concentrations in paired SD (closed circles) and LD (open circles) exposed ewes. For the SD-exposed ewe, a clear decrease during the follicular phase and increase during the luteal phase of the estrous cycle was evident. In contrast, progesterone concentrations remained below 1 ng/ml throughout the sampling period in the LD ewe. Arrow indicates time at which animals were killed.

 
Estrogen receptor immunoreactivity
Cells immunoreactive for the ER were distributed throughout the POA, bed nucleus of the stria terminalis, lateral septum, and hypothalamus (Fig. 2Go) as described previously (5, 22). Intense immunostaining was restricted to the cell nucleus, whereas faint to moderate cytoplasmic staining was noted in subpopulations of cells exhibiting strong nuclear staining in the POA and VMN (Fig. 2BGo). Nuclear and cytoplasmic immunoreactivity were both absent after the incubation of sections in adsorbed ID5 antibody.

The density of ER-immunoreactive cells within the POA differed significantly between LD and SD ewes (Fig. 3Go). Animals exposed to SD had approximately 20% fewer ER-stained cell nuclei than LD animals (P < 0.05), although single cell nuclear area measurements were not different between experimental groups (Fig. 3Go). Both SD and LD ewes exhibited cytoplasmic staining associated with nuclear immunoreactivity. The density of ER-immunoreactive cells in the VMN and ARN did not differ significantly with photoperiod, and nuclear area was similarly unchanged (Fig. 3Go).



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  		Figure 3. Quantitative analysis of the density of ER-immunoreactive nuclei (top) and the nuclear area (bottom) of these cells in the preoptic area (POA), ventrolateral division of the ventromedial nucleus (VMN), and arcuate nucleus (ARN) of ewes exposed to LD or SD photoperiod. *, P < 0.05; Student’s paired t test.

 
NPY immunoreactivity
Cell profiles immunoreactive for NPY were only located within the ARN and ME as described previously (23). Cells were small in diameter (10–15 µm), exhibited a mostly bipolar morphology and, in rostral sections, appeared to exist as a continuous cell population that flowed down from the ARN into the ME (Fig. 4AGo). Within the ME, NPY-immunoreactive neurons were scattered throughout its internal zone and exhibited a variety of morphologies (Fig. 4CGo)



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  		Figure 4. NPY immunoreactivity in the arcuate nucleus (A, B) and median eminence (C, D) of SD (A, C) and LD (B, D) -exposed ewes. Note the marked increase in the number of NPY-immunoreactive cell profiles detected in the short day animals compared with LD ewes. E and F give high-powered photomicrographs of NPY neurons (grey cytoplasmic staining) coexpressing ER immunoreactivity (black nuclei) in the arcuate nucleus (E) median eminence (F) of an SD-exposed ewe. Arrows mark ER-expressing NPY neurons. Scale bars: (A, B) 80 µm; (C, D) 40 µm; (E, F) 5 µm.

 
Exposure to SD resulted in larger numbers of NPY-immunoreactive cell profiles being detected in the ARN and ME (Figs. 4Go and 5Go). The number of NPY-immunoreactive cell profiles identified in both the rostral and caudal ME were 3- to 4-fold greater in SD animals compared with LD ewes (P < 0.05; Figs. 4Go, C and D, and 5). Although the same trend was apparent in the rostral ARN (Fig. 4Go, A and B), this failed to reach significance and, similarly, no difference was found in the caudal ARN (Fig. 5Go).



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  		Figure 5. Quantitative analysis of the number of NPY-immunoreactive cell profiles (left) and the percentage of ER-expressing NPY cells (right) in the median eminence and arcuate nucleus of LD- and SD-exposed ewes. *, P < 0.05; Student’s paired t test.

 
Double-labeling immunocytochemistry revealed relatively few NPY-immunoreactive cells in the ME that were also positive for ER (Fig. 4Go, E and F). The total number of double-labeled cells detected were small in both SD (rostral: 0.4 ± 0.2; caudal: 0.2 ± 0.1 cells/section) and LD ewes (rostral: 0; caudal: 0.2 ± 0.2) and represented only 0.1–3.0% of all NPY-immunoreactive profiles detected within the ME at both rostral and caudal levels (Fig. 5Go). Larger numbers of double-labeled cells were detected in the ARN where they were concentrated in the dorsomedial aspect of the nucleus. A clear rostro-caudal bias in the expression of ER immunoreactivity by NPY-positive cells was suggested (Fig. 5Go). Approximately 3% of all NPY-immunoreactive cells in the rostral ARN were found to express ERs (SD; 1.0 ± 0.3 double-labeled cells/section and LD; 0.8 ± 0.6) compared with approximately 10% in the caudal ARN (SD; 3.8 ± 1.4 double-labeled cells/section and LD; 2.8 ± 1.0). No effect of photoperiod was detected on double-labeled cell numbers in either the ME or ARN (Fig. 5Go).

Tyrosine hydroxylase immunoreactivity
Cells immunoreactive for TH were detected throughout the POA and hypothalamus as described previously (21). Cells comprising the A14 group were divided into those found within the periventricular area of the POA, including the OVLT (POA-A14), and those detected in the periventricular and ventral regions of the anterior hypothalamus (AHA-A14). These A14 cells usually exhibit a bipolar morphology (Fig. 6AGo). Cell count analysis found no effect of photoperiod upon the number of TH-immunoreactive cells comprising the A12, POA-A14, AHA-A14, or A15 populations (Fig. 7Go).



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  		Figure 6. High-powered photomicrographs of sections double-labeled for TH and ER in the ventral periventricular anterior hypothalamus (A, A14), arcuate nucleus (B, A12), retrochiasmatic area (C, A15), and preoptic area (D, A14). Open arrows mark TH-immunoreactive neurons without ER colocalization; small arrows indicate cells immunoreactive only for the ER, whereas large arrows in B indicate cells coexpressing TH and ER immunoreactivity. The double-labeled cells in A and D are not arrowed. Note the absence of ER staining in the A15 cells but presence of adjacent ER only positive cells. Scale bars: (A, D) 5 µm; (B, C) 10 µm.

 


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  		Figure 7. Quantitative analysis of the number of TH immunoreactive cell profiles and the percentage of ER-expressing TH neurons comprising the preoptic region of the A14 (A14-POA), anterior hypothalamic region of the A14 (A14-AHA), A12 and A15 of LD- and SD-exposed ewes.

 
Double-labeling immunocytochemistry experiments revealed TH-positive cells expressing ER immunoreactivity in the A12 (Fig. 6BGo) and A14 cell (Fig. 6Go, A and D) groups but no coexpression in the A15 (Fig. 6CGo). No ER immunoreactivity was detected in the TH cells of the lateral hypothalamus belonging to the A14 group or in the A13 cells concentrated within the dorsal hypothalamus (not shown). No significant differences in the number of TH-immunoreactive cells expressing the ER were detected with changing photoperiod (Fig. 7Go). Within the POA-A14, 13–18% of the small number of detected cells contained ERs, while up to 25% of all TH cells in the AHA-A14 were positive for the ER. Within the A12 population of the ARN, we found that approximately 15% of TH-immunoreactive cells were stained for the ER (Fig. 7Go).

ß-Endorphin immunoreactivity
Cells immunoreactive for ßE were found in the ARN only, as described previously (23, 24). Cells exhibited a variety of morphologies and were located throughout the ARN including in its most lateral extent (Fig. 8Go). Animals exposed to the SD photoperiod exhibited reduced numbers of ßE-immunoreactive cells in both the rostral and caudal ARN (Figs. 8Go and 9Go). The fall in ßE cell numbers did not quite reach the 5% significance level in either the rostral (P = 0.11) or caudal (P = 0.06) ARN, but when combined and analyzed for the whole ARN, a significant (P < 0.05) 35% reduction in ßE-immunoreactive cell number was evident in SD ewes compared with LD animals (Fig. 9Go). Insufficient pairs of SD/LD ewes underwent ER-ßE double labeling immunocytochemistry to enable statistical comparisons to be made.



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  		Figure 8. Low (A, B) and medium (C, D) powered photomicrographs of ß-endorphin immunoreactivity in the arcuate nucleus of ewes exposed to (A, C) LD and (B, D) SD. The frames in A and B indicate the enlarged area shown in C and D, respectively. Scale bars: (A, B) 80 µm; (C, D) 40 µm.

 


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  		Figure 9. Quantitative analysis of the number of ßE-immunoreactive cell profiles detected in the rostral and caudal halves of, and entire (total) arcuate nucleus of LD and SD-exposed ewes. *, P < 0.05; Student’s paired t test.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Photoperiod and estrogen receptor expression
The overwhelming evidence for changes in the sensitivity of estrogen feedback actions on the reproductive axis has led several investigators to examine the possibility that ER expression in the ovine hypothalamus may change with season (25, 26, 27). These studies have not, however, provided any clear consensus as to whether season alters ER expression; Clarke and colleagues (25) reported that cytosolic ER concentrations within the hypothalamus were elevated in anestrous ewes, whereas Glass and co-workers (26) could find no effect of season. Further, Bittman and Blaustein (27) showed that nuclear ER binding was not significantly altered by photoperiod within the POA and hypothalamus of ewes.

Using an immunocytochemical approach, which has enabled a cellular analysis of ER expression, we now report that the numbers of ER-expressing cells within the POA are elevated by around 20% in anestrous ewes compared with luteal phase animals. It is interesting to note that Bittman and Blaustein (27) identified a similar, but nonsignificant, increase in ER binding within the POA of anestrous ewes. We have taken care to compare luteal phase animals with anestrous ewes to ensure that circulating estrogen concentrations are similar (11), and we believe, therefore, that the elevation in ER immunoreactive cell numbers observed in anestrous ewes is not secondary to any influence of estrogen on ER expression (27). Although we cannot discount the possibility that the seasonal changes in ER expression within the POA may be attributable to the fluctuating progesterone profile of cycling ewes, we note that Blache and colleagues (28) were unable to find any effect of progesterone administration on ER-immunoreactive cell density in the POA. The absence of any significant changes in ER immunoreactivity in the VMN and ARN of the present study highlights the specificity of estrogen-receptive cells within the POA in terms of potential photoperiodic regulation.

The physiological significance of this change in the number of ER-expressing cells within the POA is not known. In terms of LH secretion, it seems most likely that estrogen acts indirectly upon GnRH neurons to inhibit their secretory activity during anestrus (7). Preliminary data indicate that the ER-expressing cells of the rostral POA, but not other regions, are activated by estrogen in anestrous ewes (29), and we now show that greater numbers of ER-immunoreactive cells exist in the POA at this time. Together, these observations suggest that a subset of rostral POA neurons may be induced to express ERs by an LD photoperiod and that these cells may play a role in facilitating the inhibitory effects of estrogen on GnRH neurons during anestrus. The neurochemical phenotype of the POA neurons induced to express ERs by LD photoperiod remains to be determined.

Photoperiod and tyrosine hydroxylase
A substantial body of evidence implicates dopamine as one of the neurotransmitters involved in mediating the inhibitory effect of estrogen on LH secretion in anestrous ewes. Dopamine D2 receptors are involved in the estrogen-dependent suppression of LH pulse frequency in anestrous ewes (30, 31, 32), and lesion studies suggest that this involves the A14 and/or A15 dopaminergic cell populations of the anterior hypothalamus (10, 33). Recent investigations have shown that both cell populations are activated by estrogen during anestrus but not the breeding season (34, 35). In the present study, we report that photoperiod does not influence the number of TH-immunoreactive cells detected per section in either of these dopaminergic cell populations or in the A12 cells of the ARN. This result appears to concur with others who have shown that photoperiod does not influence TH enzyme activity in the A12, A14, or A15 cells (12) or TH mRNA content in the A15 (36). Hence, in contrast to TH activity in the ME (12), photoperiod may not cause appreciable alterations in TH synthesis or activity within dopaminergic perikarya of the hypothalamus.

Because estrogen influences the A14 and A15 neurons only in anestrus, we questioned whether photoperiod may alter ER expression within these cell populations. As reported previously in ovariectomized ewes (6, 8), we were also unable to detect ER immunoreactivity within A15 cells regardless of the prevailing photoperiod. Recent evidence that the A15 neurons do not project to the ME (35, 37), where the season-dependent effects of dopamine on GnRH secretion occur (12, 38, 39), suggests that these cells may influence LH secretion indirectly. In terms of the A14 neurons, we show here that up to 25% of TH-immunoreactive cells located in the ventral periventricular region of the AHA express ERs, although this percentage does not change with season. Hence, in terms of ER immunoreactivity, we can find no evidence to support the hypothesis that changes in the level of ER expression by A14 or A15 neurons are involved in their season-dependent activation by estrogen. The basis for this phenomenon may lie instead in photoperiod-induced changes in ER-dependent transcriptional or translational events within these cells and/or the alteration of any estrogen-sensitive afferent input. In this regard, the increased numbers of ER-expressing cells identified within the POA of anestrous ewes may be of importance.

Photoperiod and NPY
We have found that SD photoperiod induces a 3-fold increase in the number of NPY-immunoreactive cells detected throughout the ME. A similar trend was observed in the rostral ARN but failed to reach statistical significance. Although the ME was not examined in a preliminary report by Yang and colleagues (16), they did provide evidence that NPY mRNA expression in the ARN is elevated in breeding season ewes compared with anestrous animals. This suggests that the increased number of NPY-immunoreactive cells observed here in breeding season ewes may result from photoperiodic influences that increase NPY synthesis. Equally, however, we cannot discount that our observations may represent an alteration in NPY transport within ME neurons and future studies will be necessary to establish whether the NPY mRNA content of neurons in the ME is also regulated by photoperiod. The mechanisms underlying the changes in NPY immunoreactivity are not established, but the low (<3%), photoperiod-independent number of NPY neurons expressing ERs in the ME makes altered efficacy of direct estrogen action on these cells unlikely. The elevated progesterone concentrations associated with reproductive activity may have a role but, again, the mode of progesterone action, if any, on NPY cells is unknown.

The role of NPY in regulating GnRH neurons in the ewe is not entirely clear. The ovine GnRH neurons receive a direct NPY input (40), but the icv or iv administration of NPY has been shown to increase (41, 42), decrease (43, 44, 45) or exert no effect (46) on LH release. The icv immunoneutralization of NPY does, however, appear to consistently abolish or delay the LH surge in breeding season ewes (42, 46). Hence, it seems likely that, as in the rat (47) and monkey (48), NPY has an excitatory influence on GnRH secretion. Our observation of a positive correlation between reproductive activity and the elevated NPY immunoreactivity in ME neurons would, therefore, be in general agreement with the hypothesis that SD photoperiod induces a change in the activity of NPY neurons that may contribute to the increased GnRH/LH release occurring during the breeding season.

Photoperiod and ß-endorphin
The involvement of endogenous opioid peptides in the regulation of GnRH secretion in the ewe is well established. The ßE neurons of the ARN appear to represent the most important opioid population, and there is good evidence that ßE can regulate GnRH secretion at the level of the terminal (24) as well as the GnRH cell body (49, 50). We report here that the number of ßE-immunoreactive neurons detected within the ARN is approximately 35% lower in luteal-phase breeding season animals compared with anestrous ewes. As is the case for our results with NPY immunoreactivity, which show an opposite pattern of photoperiod-dependent staining, we are unable to determine whether the reduced numbers of ßE cells in SD ewes results from a decrease in ßE synthesis or an increase in ßE transport away from the cell body. Recent work in the male hamster has revealed a clear suppression of POMC mRNA expression by SD (51). Although further work must been done to determine the mechanism by which photoperiod influences ßE neurons in the ewe, it remains highly likely that the ßE content of ARN cell bodies is substantially different in anestrous and luteal-phase breeding season ewes.

Previous studies suggest a seasonal variation in the role of opioid peptides in regulating LH secretion in the ewe. In anestrus, ßE is unlikely to exert any strong influence on GnRH secretion (13, 14), whereas, in the luteal phase of the breeding season, there is a consensus that opioid peptides effect the negative feedback actions of progesterone on LH secretion (14, 52, 53). Little evidence implicates ßE in the estrogen negative feedback actions of estrogen on GnRH secretion (54). The gradual increase in ßE release within the ME that occurs in concert with the rising estrogen concentrations of the follicular phase suggest a possible role for ßE in the timing of the LH surge (24). In this setting, one plausible explanation for our immunocytochemical observations would be that the elevated activity of ßE neurons in the breeding season involves increased ßE transport and release and, hence, reduced cell body ßE content and immunoreactivity as compared with anestrus.

Summary
The present study has revealed a number of neurochemical and ER immunocytochemical changes within the hypothalamus of intact ewes exposed to different photoperiods. In summary, cycling animals exposed to SD photoperiods exhibit fewer ER-expressing cells in the rostral POA, fewer ßE-immunoreactive cells within the ARN, and more NPY-positive neurons in the ME. No changes were found in TH immunoreactivity or in the density of ER-expressing cells in the VMN or ARN. Such findings highlight the potential for photoperiod to orchestrate changes in the synthetic and/or electrical activity of specific neuronal cell populations in the ovine brain.

In terms of understanding the mechanisms through which estrogen regulates GnRH neurons in the different seasons, our most important observation is that the LD photoperiod appears to induce ER expression within a subpopulation of rostral POA cells. We speculate that these cells may be involved in mediating part of the negative feedback effects of estrogen on GnRH neurons during anestrus. Although we have been able to demonstrate relatively high ER expression in ventral hypothalamic A14 dopaminergic neurons and the presence of ERs in NPY neurons, we have found no significant effect of photoperiod on the ability of these cells to display ER immunoreactivity. Together, these data suggest that only specific estrogen-receptive neuronal cell populations, such as those of the POA, may be influenced by photoperiod to alter their level of ER expression. By identifying substantial changes in NPY and ßE immunoreactivity, this study provides an insight into which neuronal cell populations may be functioning differently in intact SD- and LD-exposed ewes. Future studies will now be required to establish whether such changes result from a direct action of photoperiod or are secondary to the different gonadal steroid profiles characteristic of anestrous and cycling ewes.


   Acknowledgments
 
We thank G. Delsol for his generous gift of the ID5 antibody, A. Dady for looking after the sheep, and I. King for histological support. Drs. B. Malpaux, J. Bicknell and J. Robinson are thanked for constructive comments on this paper.


   Footnotes
 
1 St. Catharine’s College Research Fellow. Present address: INRA, Physiologie de la Reproduction des Mammifères Domestiques, Nouzilly, 37380 France. Back

2 Lister Institute-Jenner Fellow. Back

Received December 31, 1996.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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