Rostral Anterior Hypothalamic

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Aug 3, 2011 - neuron-specific enolase antibody (MAB377; Millipore, Billerica, MA) diluted ..... cells and the percentage of GnRH neurons exhibiting GnIH-.
BIOLOGY OF REPRODUCTION 85, 1057–1065 (2011) Published online before print 3 August 2011. DOI 10.1095/biolreprod.111.092031

Neurons of the Lateral Preoptic Area/Rostral Anterior Hypothalamic Area Are Required for Photoperiodic Inhibition of Estrous Cyclicity in Sheep1 Stanley M. Hileman,2,3 Christina J. McManus,3 Robert L. Goodman,3 and Heiko T. Jansen4 Department of Physiology and Pharmacology,3 West Virginia University, Morgantown, West Virginia Department of Veterinary and Comparative Anatomy, Pharmacology, and Physiology,4 Washington State University, Pullman, Washington INTRODUCTION

Photoperiod determines the timing of reproductive activity in many species, yet the neural pathways whereby day length is transduced to a signal influencing gonadotropin-releasing hormone (GnRH) release are not fully understood. Physical lesions of the lateral preoptic area (lPOA)/rostral anterior hypothalamic area (rAHA) in female sheep extend the period of estrous cyclicity during inhibitory photoperiods. In the present study we sought to determine whether destroying only neurons and not fibers of passage in this area would lead to similar resistance to photosuppression. Additionally, neural tract-tracing was used to map connectivity between the lPOA/ rAHA and other hypothalamic areas implicated in photoperiodic regulation of reproduction. Progesterone secretion was monitored in six sheep to determine estrous cycles for 90 days during a short-day (permissive) photoperiod. Three sheep then received bilateral injections of the excitotoxic glutamate analog, nmethyl-aspartic acid, directed toward the lPOA/rAHA, whereas three others served as controls. All were then exposed to a longday (suppressive) photoperiod for 120 days. Control sheep ceased cycling at 40 6 10 days (mean 6 SEM), whereas lesioned sheep continued cycling through the end of the study. The results of the tract-tracing study revealed both afferent and efferent projections to the medial POA, retrochiasmatic area, arcuate nucleus, and premammillary region. Furthermore, close proximal associations with GnRH neurons from efferent projections were observed. We conclude that neurons located within the lPOA/rAHA are important for timing cessation of estrous cycles during photosuppression and that this area communicates directly with GnRH neurons and other hypothalamic areas involved in the photoperiodic regulation of reproduction.

Changes in day length (photoperiod) regulate gonadotropin secretion in vertebrates as diverse as fish and primates so that reproductive activity is limited to a specific season [1]. In species like sheep that have autumnal breeding seasons, a shift from a short day length to longer day length (e.g., from 10L:14D to 16L:8D) decreases gonadotropin secretion and causes ovarian cycles to cease. In contrast, a shift from long to short days results in increased gonadotropin secretion and onset of ovarian cycles [2]. Furthermore, if animals are maintained on constant photoperiods for a prolonged period (e.g., 150 days), they undergo a spontaneous reversal in gonadotropin secretion and reproductive activity, a phenomenon known as photorefractoriness [3]. Finally, these photoperiod-induced effects interact with an endogenous circannual oscillator to determine the timing and duration of the breeding season [4]. Day length is converted into a neural signal in the brain via the pineal gland hormone, melatonin. Melatonin is released nocturnally [2], thus duration of melatonin release is longer in short days (long nights) than in long days (short nights). The photoperiod (melatonin) response is reflected in a change in responsiveness to estradiol-negative feedback that in turn alters release of gonadotropin-releasing hormone (GnRH), and thus luteinizing hormone (LH). Only fragmentary information currently exists about the anatomical sites of action for melatonin and which neurochemical systems it uses to alter GnRH secretion, although there does not appear to be a direct effect on the GnRH neuron [5]. A potential site through which melatonin may act to mediate the effects of photoperiod (in particular, photosuppression) is the lateral preoptic area (lPOA)/rostral anterior hypothalamic area (rAHA). Melatonin binding has been demonstrated in this region [6], and lesions of the AHA block the inhibitory effects of melatonin on blastocyst implantation in the skunk [7]. Furthermore, lesions of the AHA render hamsters unresponsive to short-day photoperiods, which are inhibitory in this species [8]. In sheep, we previously observed that radiofrequency lesions of the lPOA/rAHA inhibited the ability of a long-day photoperiod to suppress reproduction [9]. A limitation of this previous work was that the electrolytic lesions could have ablated not only neurons within the lPOA/rAHA, but neural fibers of passage as well. To circumvent this issue, the current study used n-methyl-aspartic acid (NMA) to induce excitotoxic lesions [10–12] and test the hypothesis that cells within the lPOA/rAHA are functionally important for control of seasonal breeding. Once these lesions were confirmed to be effective, we then performed a second study to test the hypothesis that the lPOA/rAHA is anatomically linked with other brain regions implicated in the photoperiodic control of reproduction, in particular the medial POA (mPOA), suprachiasmatic nucleus (SCN), retrochiasmatic area (RCH), arcuate nucleus (ARC), and premammillary region (PMR).

estrous cycle, neuroendocrinology, ovine/sheep, photoperiod, seasonal reproduction

1 Supported by US Department of Agriculture grants 01-10835 to S.M.H. and 05-15848 to H.T.J. Image analyses were performed in the West Virginia University Imaging Facility, which is supported in part by the Mary Babb Randolph Cancer Center and National Institutes of Health grant P20 RR016440. 2 Correspondence: Stanley M. Hileman, Department of Physiology and Pharmacology, West Virginia University, P.O. Box 9229, 3047 Health Sciences North, Morgantown, WV 26506. FAX: 304 293 3850; e-mail: [email protected]

Received: 28 February 2011. First decision: 27 March 2011. Accepted: 13 July 2011. Ó 2011 by the Society for the Study of Reproduction, Inc. eISSN: 1529-7268 ISSN: 0006-3363


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FIG. 1. Photomicrograph of representative staining for neuron-specific enolase in a ewe that had received no injection (A and B) and 0.05 lg (1 lg/ll in a volume of 50 nl) of NMA (C and D) injected unilaterally into the lPOA/rAHA. A and C are at 43 magnification, and B and D are a 403 magnification view of a control and lesioned area. Arrow denotes example of a positively stained neuron. Dotted lines in C show the lateral extent of the lesion. Bars ¼ 250 lm (A and C) and 25 lm (B and D).

All procedures described in the present study were performed with the approval of the animal care and use committees at West Virginia University and Washington State University and were in accordance with the National Institutes of Health’s ‘‘Guide for the Care and Use of Laboratory Animals.’’ Before surgeries, animals were kept outdoors. Just prior to and after surgeries, animals were kept in either light-sealed photochambers (experiment 1) or in rooms with lighting adjusted to mimic natural day length (experiment 2). Temperature was maintained between 158C and 238C, and ewes were maintained on alfalfa pellets supplemented with corn and had free access to water.

Experiment 1: Effect of Excitotoxic Lesions on Estrous Cyclicity under an Inhibitory Long-Day Photoperiod Neurotoxic lesions of the lPOA/rAHA were accomplished by stereotaxically placed injections of NMA [10–12]. A preliminary study (n ¼ 2) was performed to determine the dose of NMA that would produce a lesion of approximately the same size as our earlier electrolytic lesions [9]. Female sheep were injected unilaterally with NMA at a dose of 1 lg/ll in volumes of 25 nl (n ¼ 1) or 50 nl (n ¼ 1) using stereotaxic procedures described below. Seven days later, sheep were killed with an overdose of pentobarbital, brains were perfused with paraformaldehyde, hypothalamic tissue was collected, 50-lm-thick frozen coronal sections were cut, and a set of these were processed for immunocytochemistry as previously described [9]. Assessment of lesion size was made by staining for neuron-specific enolase and visually comparing the area devoid of neural staining in NMA-lesioned ewes with that from our previous work (Fig. 1) [9]. After determining that the 50 nl volume of NMA produced a lesion of the appropriate size, six adult, ovary-intact sheep of mixed breeding were placed in photoperiod-controlled rooms on a short-day photoperiod (7L:17D) for 90 days. After approximately 85 days of exposure to the short-day photoperiod, sheep either received stereotaxic injections of NMA (50 nl; n ¼ 3), underwent sham surgery (n ¼ 1), or did not undergo surgery (n ¼ 2). Following surgery, sheep were switched to a long-day photoperiod (16L:8D) and maintained on this for 120 days. Blood samples were collected twice weekly via jugular venipuncture during the course of the experiment and were assayed for progesterone to identify estrous cycles. At the end of the study, hypothalami were collected and assessed for lesion placement and size as described above.

Experiment 2: Identification of lPOA/rAHA Connectivity with other Brain Regions Important for Seasonal Breeding Because results of the first study revealed that cells within the lPOA/rAHA were important for timing the photoperiod-induced suppression of estrous

cycles, a second study was performed to determine whether this brain region was neuroanatomically linked to other brain regions implicated in the photoperiodic control of reproduction. To this end, female sheep received injections of either a combination (n ¼ 3) of the anterograde tract-tracing agent biotinylated dextran amine (BDA; 10% wt/vol) and the retrograde tract-tracing agent cholera toxin b-subunit (CTb; 0.5% wt/vol), or CTb alone (n ¼ 2). Coordinates for the injections were identical to those used in the first experiment. Two weeks after injection the sheep were euthanized with an overdose of pentobarbital, and hypothalami were collected. Neurosurgeries. Surgeries were performed as previously described [13, 14]. Briefly, using sterile techniques, female sheep were anesthetized with halothane (approximately 2%) and placed in a stereotaxic frame (David Kopf Instruments, Tujunga, CA). After the skull was exposed, a 20-mm-wide 3 30mm-long hole, centered about 10 mm rostral to bregma, was drilled, and the skull was removed to allow the superior sagittal sinus to be ligated. A sharpened 18-G stainless steel tube was lowered into the lateral ventricle just rostral to bregma and 4 mm lateral to midline, and then 2 ml of radio-opaque dye (iohexol; Omnipaque 350; Winthrop, New York, NY) was injected over a 1-min period to allow visualization of the ventricles and for target coordinate determination. For injection placement, 18-G sharpened stainless steel guide tubes (length: 57 mm) were lowered bilaterally to a position 2 mm above the target sites for microinjection of NMA (2 mm lateral to midline, 2 mm dorsal to the floor of the optic recess of the third ventricle, 1 mm posterior to the rostral wall of the third ventricle). Hamilton syringes (1 ll; model 7101; Hamilton, Reno, NV) were then inserted into the guide tubes, and 50 nl of NMA (1 lg/ll) was injected bilaterally over 5 min (except for the one ewe in the preliminary study that received 25 nl). The syringe was left in place after injection for 5 min prior to removal. For tract tracing, a single 18-G guide tube was lowered using the coordinates described above. A Hamilton syringe was lowered to the final coordinates described above, and a single injection was made of either the retrograde tracing agent CTb (200 nl), the anterograde tracing agent BDA (200 nl), or a combination of the two (200 nl each). Injections took place over 5 min, with the needle left in place for an additional 5 min before withdrawal. Following needle withdrawal, the guide cannula(e) and the lateral cannula were removed, the exposed brain was covered with gelfoam, and skin was sutured. Dexamethasone was administered i.m. in decreasing daily doses as an antiinflammatory agent and to prevent swelling, beginning with 20 mg on the day prior to surgery and ending 3 days afterward with 2 mg. Penicillin (6 ml) was also injected daily during this period, and atropine (15 mg) was given immediately prior to surgery. An analgesic (flunixin meglumine; 100 mg) was administered immediately prior to surgery. Tissue collection, verification of lesion sites, and immunocytochemistry. Tissue collection was performed as described previously [15]. Specifically, sodium heparin (20 000 IU) was injected i.v. 10 min before, and again immediately prior to, the administration of a lethal dose of sodium pentobarbital (about 2000 mg i.v.). The head was quickly removed and perfused via both internal carotid arteries with 6 L of 4% paraformaldehyde in

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RESULTS Experiment 1 Comparison of the control and NMA-injected side of the lPOA/rAHA in a female sheep injected with 50 nl of NMA can be seen in Figure 1. Staining for neuron-specific enolase showed abundant neuronal nuclei in the uninjected side (Fig. 1, A and B). In contrast, NMA almost totally eliminated staining in the area surrounding the injection site (Fig. 1, C and D). The area covered by the NMA-induced lesions within the lPOA/ rAHA of each animal is shown in Figure 2. Both location and area covered by these neurochemical lesions were very similar to those of our previous study [9]. Lesions were bilateral and confined to the lPOA/rAHA.

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0.1 M PO4 buffer containing 1.0 IU/ml sodium heparin and 0.1% NaNO3 (a vasodilator). The brains were removed, and tissue blocks containing the diencephalon were dissected out and stored at 48C in this fixative overnight, and then transferred to 0.1 M phosphate buffer containing 20% sucrose until they sank. Frozen coronal sections (50 lm thick) were cut on a freezing microtome into five series. Every fifth section was analyzed for neuron-specific enolase. Immunocytochemical identification of neuron-specific enolase was performed on free-floating sections [16]. On Day 1, sections were washed 3 3 20 min in 0.1 M PBS, followed by a 10-min incubation in 1% H2O2. Tissue was then washed 3 3 5 min in PBS, placed in 4% normal donkey serum (NDkS) with 0.1% Triton X for 1 h, and then placed overnight on a shaker table in antineuron-specific enolase antibody (MAB377; Millipore, Billerica, MA) diluted 1:1000 in PBS with 4% NDkS and 0.1% Triton X. On Day 2, sections were washed 3 3 5 min in PBS, followed by 1 h of incubation in 1:400 biotinylated donkey anti-mouse immunoglobulin G (Vector Laboratories, Burlingame, CA) in 4% NDkS and 0.1% Triton X. Tissue was then washed 3 3 5 min in PBS, followed by a 1-h incubation in ABC diluted 1:400 (Vector Laboratories) in PBS. After 3 3 5 min washes in PBS, sections were incubated for 10 min in nickel-enhanced diaminobenzidine (DAB) [17], washed again 3 3 5 min in PBS, mounted on slides, and dried overnight. On Day 3, sections were placed in acetate buffer for 30 sec, stained in 0.5% cresyl violet for 15 min, dehydrated through a series of alcohols, cleared in xylene, and then coverslipped using Permount (Fisher Scientific, Pittsburgh, PA). This procedure produces a blueblack reaction product that is constrained to the nucleus. Lesion extent was determined by identifying areas devoid of nuclear staining (Fig. 1) when viewed at the light microscope level. Biotinylated dextran amine and CTb were visualized as previously described [18]. For BDA, sections were washed 3 3 5 min in PBS, incubated for 10 min in 1% H2O2, washed again in PBS, and then incubated for 1 h in ABC diluted 1:400 in PBS. After 3 3 5 min washes in PBS, sections were incubated for 10 min in nickel-enhanced DAB. For CTb visualization on the BDA-labeled sections, tissues were then washed 3 3 5 min in PBS, followed by incubation for 1 h in PBS containing 0.4% Triton X and 4% normal goat serum for 1 h. Tissues were then washed and incubated overnight at 48C on a shaker table in anti-rabbit CTb (C-3062; Sigma-Aldrich, St. Louis, MO) diluted 1:15 000 in PBS containing 0.4% Triton X. The next day, sections were washed and then incubated for 1 h in goat anti-rabbit immunoglobulin G (1:200; Vector Laboratories). Tissue was then washed 3 3 5 min in PBS, followed by a 1-h incubation in ABC diluted 1:400 in PBS. After 3 3 5 min washes in PBS, sections were incubated for 10 min in DAB [17], washed again for 3 3 5 min in PBS, mounted on slides, and dried overnight. Tissues were then dehydrated through a series of alcohols, cleared in xylene, and coverslipped using Permount. For mPOA tissue where GnRH was visualized in addition to BDA, tissues were incubated as described above except that mouse anti-GnRH (SMI41; lot 3; Sternberger Monoclonals, Covance Inc., Princeton, NJ) was used as the primary antibody at 1:3000. Measurement of progesterone. Progesterone concentrations in plasma were assessed by using a radioimmunoassay kit (Diagnostic Products Corp., Los Angeles, CA). Sensitivity, as defined by 90% B/Bo, was 0.5 ng/ml. All samples were run in a single assay, with the intraassay coefficient of variation being 6.4%. Statistics. Time to cessation of estrous cycle expression was defined to be the time from the beginning of the long-day photoregimen to the first of four consecutive blood samples (i.e., a period of 2 wk) that contained less than 1 ng/ ml progesterone. We originally had planned to compare time to cessation of estrous cycles using a Student t-test. However, because none of the lesioned animals became anestrous, there was no variability associated with this treatment group. Thus, only means for each group are presented.


FIG. 2. Schematic depicting the location of NMA-induced lesions. Lesioned areas, as determined by the absence of staining for neuronspecific enolase, are depicted by the black areas. AC, anterior commissure; FX, fornix; OC, optic chiasm; SON, supraoptic nucleus.

Profiles of progesterone secretion for all animals are shown in Figure 3. All six sheep exhibited a normal pattern of estrous cycles during the short-day photoperiod. After placement in the long-day photoperiod, nonsurgical controls and the single sham-operated sheep showed only two complete estrous cycles, with a mean period to cycle cessation (anestrus) of 40 6 10 days. In contrast, NMA-injected ewes exhibited five to seven estrous cycles following the switch to the long-day photoperiod. Indeed, by our definition for cessation of estrous cycle expression, none of the three lesioned sheep had entered anestrus by the end of the experiment. Experiment 2 Schematics of the resultant CTb or BDA staining following tract-tracer injections are shown in Figures 4 and 5,



FIG. 3. Progesterone profiles for three ewes (Control) receiving either a sham surgery (K54) or no surgery (K13, K46), and three ewes receiving 0.05 lg (1 lg/ll in a volume of 50 nl) of NMA injected bilaterally into the lPOA/rAHA (NMA-lesioned). Ewes were exposed to 120 days of long-day photoperiod following surgery.

less in the region of the OVLT (Fig. 6A). A few fibers also were observed in the SCN (Fig. 6B). Fibers were evident in the RCH (Fig. 6C), and intermediate to heavy fiber densities were observed in the dorsomedial and lateral hypothalamus, with comparatively fewer projections to the ARC and ventromedial nucleus (Fig. 6D). However, a heavy concentration of fibers was evident in the ventrolateral hypothalamus. Intermediate to heavy projections were observed in the PMR (Fig. 6E). As with the distribution of CTb-labeled cells, BDA fibers were observed extending along the base of the hypothalamus, with fibers clearly evident in the amygdala. Based on the high density of fibers in the POA, we also evaluated the possibility that BDA projections onto GnRH neurons were present. Two sections from one ewe containing the mPOA at the level of the OVLT were examined. As shown in Figure 7, close proximal associations were observed between GnRH neurons and BDAlabeled fibers. Of 60 identified GnRH neurons, 27 (45%) appeared to have BDA-containing close proximal associations. DISCUSSION Control of seasonal reproduction by photoperiod involves a complex interplay between various areas near or in the hypothalamus, such as the PMR, RCH, ARC, and POA, with hormones, such as estradiol, melatonin, and thyroid hormones [19–21]. The work presented in the present study supports the hypothesis that an additional area, the lPOA/rAHA, is an important component of the circuitry of seasonal neuroendocrine control. Specifically, we demonstrate that following ablation of cells within this region, female sheep exhibit resistance to the inhibitory effects of long-day photoperiods on ovarian cycles. Furthermore, although our tract-tracing results raise the possibility of cells within the lPOA/rAHA directly influencing GnRH neurons, they also clearly show that this

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respectively. The injection site locations were very similar to those of the excitotoxic lesions induced in experiment 1 and in our previous work. Importantly, injection of these amounts of tract-tracing agents resulted in an area of injection that was similar in scope to the area lesioned in this and our previous study. Cholera toxin b-subunit-labeled cells were found throughout the POA and the extent of the hypothalamus in an area generally bordered laterally by the fornix and mammillothalamic tracts. Injections were similarly placed in all three sheep, and a tracing from a representative sheep is shown in Figures 4 and 5. Retrogradely labeled cells (Fig. 4) were largely observed on the side that was ipsilateral to the injection, with only scattered cells observed on the contralateral side. We observed CTb-labeled cells in several areas known to be important to the photoperiodic regulation of reproduction. Numerous cells were observed in the mPOA, although they were less abundant in the organum vasculosum lateral terminalis (OVLT; Fig. 6A). We also observed scattered cells in the SCN (Fig. 6B) and the RCH just medial to the optic tracts (Fig. 6C). There were also a large number of cells located within the mediobasal hypothalamus, namely the ARC, dorsomedial nucleus, and lateral hypothalamus, but relatively fewer cells in the ventromedial nucleus (Fig. 6D). A high number of CTb-labeled cells were noted in the ventrolateral hypothalamus, and a relatively high density of cells was also observed in the PMR (Fig. 6, E, higher magnification in F). There were also scattered cells extending laterally along the base of the hypothalamus, and cells were also observed in the area of the amygdala. Although not shown, the distribution of cells in ewes receiving only CTb was virtually identical to that of ewes receiving both CTb and BDA. Examination of BDA staining (Fig. 5) revealed a pattern that was quite similar to that of CTb staining. A heavy concentration of fibers was seen in the POA, with comparably



FIG. 4. Tracings of cells positive for CTb in sections from the POA through the hypothalamus of a ewe that had received a unilateral injection of the retrograde tracing agent CTb (200 nl) and the anterograde tracing agent BDA (200 nl). 3V, third ventricle; AC, anterior commissure; AMG, amygdala; DMH, dorsomedial hypothalamus; FX, fornix; INJ, injection site; MB, mammillary body; MT, mammillothalamic tract; OC, optic chiasm; OT, optic tract; PD, pars distalis; PT, pars tuberalis; SON, supraoptic nucleus; ST, stria terminalis; VMH, ventromedial hypothalamus.

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FIG. 5. Tracings of nerve fibers positive for BDA in sections from the POA through the hypothalamus of a ewe that had received a unilateral injection of the retrograde tracing agent CTb (200 nl) and the anterograde tracing agent BDA (200 nl). The sections shown here are from the same ewe as in Figure 4. Nerve fiber density: H, high; I, intermediate; L, light; VL, very light. AC, anterior commissure; AMG, amygdala; DMH, dorsomedial hypothalamus; FX, fornix; INJ, injection site; MB, mammillary body; MT, mammillothalamic tract; OC, optic chiasm; OT, optic tract; PD, pars distalis; PT, pars tuberalis; SON, supraoptic nucleus; ST, stria terminalis; 3V, third ventricle; VMH, ventromedial hypothalamus.

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FIG. 6. Photomicrographs (original magnification 310) of cells positive for CTb and nerve fibers positive for BDA in the POA (A), SCN (B), RCH (C), mediobasal hypothalamus (D), and PMR of the hypothalamus (E). A 403 magnification of cells (asterisk) and fibers (arrow) in the PMR is shown in F. 3V, third ventricle; DMH, dorsomedial hypothalamus; OC, optic chiasm; OT, optic tract; VMH, ventromedial hypothalamus. Bars ¼ 200 lm (A–E) and 50 lm (F).

these associations were identified at only the light microscope level in one animal, and evaluation of the extent to which GnRH neurons may be contacted is limited by the fact that tract-tracing injections were only done on one side of the

FIG. 7. Photomicrograph of two GnRH neurons (brown cytoplasmic staining) from the mPOA at the level of the organum vasculosum lamina terminalis exhibiting close proximal associations from nerve fibers stained for BDA (black) following injection of BDA into the lPOA/rAHA. Original magnification 3100; bar ¼ 30 lm (A) and 60 lm (B).

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region is anatomically connected with other areas already implicated in mediating the effects of photoperiod on reproduction. Thus, the reproductive outcomes observed in our study likely involve not only neurons of the lPOA/rAHA, but those in other regions as well. Our previous work showed that radiofrequency lesions placed in the lPOA/rAHA extended the period of estrous cycles following exposure to long-day photoperiods [9]. Interestingly, these lesions had no influence on the timing of estrous cycle onset following exposure to short-day photoperiods, and effects persisted as similar responses were exhibited through two complete short-day/long-day photoperiod cycles [9]. The present study builds on that initial study through the use of NMA as a lesioning agent. This has the advantage of selectively destroying cell bodies without damaging neural fibers of passage [10–12], a critical distinction that we could not make previously. Similar to our earlier study, a profound resistance to the inhibitory effects of a long-day photoperiod occurred in lesioned ewes. Taken together, these studies clearly indicate that neuronal cell bodies in the lPOA/rAHA play a role in mediating the inhibitory effects of long days. Although the results from this study indicate that the lPOA/ rAHA is involved in photoperiodic regulation of reproduction, the precise mechanism whereby this occurs is not clear. We have now identified GnRH afferents from this region, which raises the possibility of direct control of GnRH release. However, this finding must be interpreted with caution because



coexpress kisspeptin, neurokinin B, and dynorphin (termed KNDy neurons). KNDy neurons express ESR1 [36], and kisspeptin is a potent stimulator of GnRH/LH release in several species [37–40]. Expression of kisspeptin is inhibited by gonadal steroids [36, 41] and is suppressed during an inhibitory photoperiod [42, 43]. These data support an important role for kisspeptin in the seasonal regulation of reproduction. In addition, we have recently shown that NKB stimulates LH release during the follicular phase of the estrous cycle [44] and in peripubertal female ewe lambs (Hileman and Goodman, unpublished results). With regard to the dorsomedial hypothalamus, cells in this area express the RFamide-related peptide, gonadotropin-inhibiting hormone (GnIH) [42, 45]. Although the role of GnIH in seasonal reproduction is not entirely clear for sheep [46], the number of GnIH-containing cells and the percentage of GnRH neurons exhibiting GnIHcontaining close contacts are lower during the breeding season than nonbreeding season [42], consistent with its role as an inhibitor of GnRH/LH secretion [46]. Thus, reciprocal contacts between these areas and the lPOA/rAHA could potentially be important for regulating the response to long-day photoperiods. We observed dense reciprocal innervations of the PMR, an area that appears to be very important in the actions of both melatonin and thyroxine (T4). T4 implants in the PMR were effective in inducing anestrus in ewes [47], and melatonin implants in this site (presumably providing a short-day signal) during long days induced LH secretion [48]. Further, an area of high melatonin binding in the caudal ARC/PMR [48] corresponds very well with an area receiving a high density of BDA-labeled fibers in our study. Interestingly, the responses observed in our female sheep are somewhat similar to those observed in the absence of thyroid hormones at the end of the breeding season [20] (i.e., animals do not enter anestrus). Our previous study [9] showed that lesions of the lPOA/rAHA do not disrupt circulating T4 levels or melatonin secretory patterns. Thus, input from the lPOA/rAHA may influence the response to T4 and/or melatonin, and therefore affect the timing of anestrus in response to long-day photoperiods. The data presented in the present study clearly identify the lPOA/rAHA as playing a role in the photoperiodic control of reproduction in the sheep. Neurotoxic lesions in this region clearly interfered with the ability of a long-day photoperiod to induce anestrus in ewes. Furthermore, our tract-tracing results unequivocally show that the lPOA/rAHA is intimately connected with several regions of the hypothalamus that are involved in regulating seasonal reproduction. Additional studies will be required to identify the specific neural substrates involved and the functional relationship of this area with other photoperiod-sensitive regions of the hypothalamus. REFERENCES 1. Gwinner E. Circannual Rhythms. New York: Springer-Verlag; 1986. 2. Bittman EL, Karsch FJ, Hopkins JW. Role of the pineal gland in ovine photoperiodism: regulation of seasonal breeding and negative feedback effects of estradiol upon luteinizing hormone secretion. Endocrinology 1983; 113: 329–336. 3. Malpaux B, Moenter SM, Wayne NL, Woodfill CJ, Karsch FJ. Reproductive refractoriness of the ewe to inhibitory photoperiod is not caused by alteration of the circadian secretion of melatonin. Neuroendocrinology 1988; 48:264–270. 4. Robinson JE, Wayne NL, Karsch FJ. Refractoriness to inhibitory day lengths initiates the breeding season of the Suffolk ewe. Biol Reprod 1985; 32:1024–1030. 5. Malpaux B, Viguie C, Skinner DC, Thiery JC, Chemineau P. Control of the circannual rhythm of reproduction by melatonin in the ewe. Brain Res Bull 1997; 44:431–438. 6. Bittman EL, Weaver DR. The distribution of melatonin binding sites in neuroendocrine tissues of the ewe. Biol Reprod 1990; 43:986–993.

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lPOA/rAHA. Interestingly, cells in the lPOA/rAHA bind melatonin [6] and express estrogen receptor-alpha (ESR1) [22, 23], raising the possibility that these hormones act locally on cells within the lPOA/rAHA to influence GnRH release. Although we did not identify the phenotype of such cells within the lPOA/rAHA, neurons expressing tyrosine hydroxylase [24, 25] and dynorphin [26] in this area have been reported in the sheep. The tyrosine hydroxylase-containing neurons are likely part of the A14 dopamine cell group, and limited data in the sheep are consistent with a role for these neurons in the photosuppression of LH secretion [25, 27]. With regard to dynorphin, neurons expressing preprodynorphin mRNA are localized to the AHA, and GnRH neurons receive synaptic input from dynorphin neurons [15]. Preprodynorphin mRNA expression in the AHA is decreased by ovariectomy and restored by progesterone treatment in ewes [28], and is increased during a long-day photoperiod in rams by testosterone [29]. An additional population of dynorphin neurons in the ARC also plays a role in regulating GnRH/LH secretion [15, 26], but it is not known whether they are involved in responsiveness to long-day photoperiods. As mentioned above, control of seasonal breeding undoubtedly involves a complex interaction of a number of different areas in or near the hypothalamus with the GnRH system [21, 30]. Our tract-tracing results now reveal the connectivity of the lPOA/rAHA with several areas of known importance to seasonal breeding. Heavy innervations and high numbers of labeled cells were found in the mPOA. This is not entirely surprising because this region lies very close to the injection site; thus, some caution is required in interpreting this result. In sheep, this area contains the majority of GnRH cell bodies and a high number of ESR1-containing neurons [22–24, 31]. Indeed, previous work showed that estradiol implants in the ventral mPOA, an area just below the region of our lesions, suppressed LH in ovariectomized ewes during a long-day photoperiod [13]. Interestingly, melatonin implants placed in this region, which should provide a short-day signal, did not reverse long-day photosuppression in ewes [32], suggesting that this is not an area responsible for initiation of GnRH/LH secretion in response to stimulatory photoperiods. However, this does not rule out a role for this area in melatonin-driven changes in GnRH/LH secretion during photosuppression. Another area of note that received reciprocal input was the RCH. The RCH region contains the A15 group of dopamine neurons, and there is a large amount of evidence that these neurons, located just medial to the optic tract, are involved in mediating steroid-induced suppression of GnRH secretion during inhibitory photoperiods. Lesions of A15 dopamine neurons significantly compromise the ability of estradiol to suppress LH secretion in ewes during the nonbreeding season, but not during the breeding season [27, 33]. Furthermore, ovariectomized ewes treated with estrogen exhibit an increase in the number of A15 dopamine cells expressing the immediate early gene product FOS, a marker of neuronal stimulation, only during the nonbreeding season [25]. Finally, estradiol implants placed directly into the RCH of the hypothalamus reduce LH pulse frequency during the nonbreeding season via ESR1, but not ESR2 [14, 34]. Interestingly, A15 dopamine neurons do not express ESR1 [24, 35], so estradiol likely influences these neurons indirectly. The identities of these indirect inputs are not completely known but may include ESR1-containing neurons just dorsal to the A15 neurons or the ventral mPOA. A large number of labeled cells and BDA-labeled fibers were also observed within the mediobasal hypothalamus in the areas of the ARC and dorsomedial nucleus. The role of the ARC in regulating GnRH/LH secretion recently has received attention because of a subset of neurons residing therein that


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