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Endocrinology Vol. 140, No. 12 5929-5936
Copyright © 1999 by The Endocrine Society

ARTICLES

A Subset of Gonadotropin-Releasing Hormone Neurons in the Ovine Medial Basal Hypothalamus Is Activated during Increased Pulsatile Luteinizing Hormone Secretion1

Rachid Boukhliq2, Robert L. Goodman, Sandra J. Berriman, Brian Adrian3 and Michael N. Lehman

Department of Physiology, West Virginia University Health Sciences Center (R.B., R.L.G., B.A.), Morgantown, West Virginia 26506-9229; and Department of Cell Biology, Neurobiology, and Anatomy, University of Cincinnati College of Medicine (S.J.B., M.N.L.), Cincinnati, Ohio 45267-0521

Address all correspondence and requests for reprints to: Dr. Robert L. Goodman, Department of Physiology, West Virginia University Health Sciences Center, P. O. Box 9229, Morgantown West Virginia 26506-9229. E-mail: rgoodman{at}wvu.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
GnRH neurons active in the preovulatory LH surge have been identified in several species using the early intermediate gene product, Fos, but the GnRH neurons active during episodic LH secretion remain unknown. In this study, we have used Fos and Fos-related antigens (FRA) to determine whether a subset of GnRH neurons is active when pulsatile LH secretion is acutely stimulated in sheep. In experiment 1, episodic LH secretion was stimulated in five of six ewes by injection of an opioid antagonist to luteal phase ewes. These five ewes had a 6-fold increase in the percentage of GnRH neurons in the medial basal hypothalamus (MBH) expressing Fos/FRA, compared with control ewes that had no LH pulses before death. Fos/FRA expression was not increased in GnRH neurons found in any other area. In experiment 2, episodic LH secretion was induced in rams by introduction of estrous ewes. This treatment increased Fos/FRA expression in MBH GnRH neurons approximately 10-fold compared with control rams. Again, this increase in Fos/FRA expression in GnRH neurons was limited to the MBH. This selective activation of MBH GnRH neurons could reflect the preferential inhibition of these perikarya by endogenous opioid peptides. It also raises the possibility that a subset of GnRH neurons in the MBH may be responsible for episodic GnRH secretion in sheep.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IT is now generally agreed that secretion of LH during the normal ovarian cycle consists of two distinct components: 1) the preovulatory LH surge that occurs just before ovulation and 2) tonic LH secretion that occurs at low levels throughout the rest of the ovarian cycle. Tonic LH secretion occurs in normal males, but the LH surge does not. This concept was originally developed based on work in the rat (1, 2) and has since been extended to a wide variety of species, including the sheep (3), rhesus monkey (4), and human (4). In most spontaneous ovulators, the LH surge is induced by high levels of estradiol, while tonic LH secretion is controlled by the negative feedback actions of gonadal steroids (1, 2, 3, 4). Tonic LH secretion occurs in a pulsatile pattern (3, 4, 5) in response to the episodic release of GnRH into the hypophyseal portal circulation (5). This pulsatile pattern of GnRH release is critical for normal secretion of gonadotropins; release of these hormones from the anterior pituitary cannot be maintained by continuous infusions of GnRH (6). In addition, changes in episodic release of GnRH appear to play a critical role in inhibiting reproductive function before puberty and during seasonal anestrus (3).

The concept that different mechanisms control the preovulatory LH surge and tonic, pulsatile, LH secretion led to studies that attempted to identify specific GnRH neurons responsible for each. Early work in the rat, using knife cuts between the preoptic area (POA) and the medial basal hypothalamus (MBH), led to the proposal that GnRH perikarya in the POA are needed for the LH surge, while those in the MBH control LH pulses (2, 7, 8). However, several immunocytochemical studies have questioned the latter conclusion because GnRH perikarya have been difficult to find in the rat MBH, and the deafferentation procedures may have missed subchiasmatic projections from the POA to the median eminence (9, 10). Similar deafferentation studies in the rhesus monkey (11) and sheep (12, 13) also failed to distinguish between GnRH neurons controlling these two types of secretion. In these species, the MBH contains GnRH perikarya (10, 14, 15), and knife cuts either had no effect on the LH surge or pulsatile LH secretion (the monkey) or decreased the amplitude of both (the sheep). Spikes in multiunit electrical activity have been associated with LH pulses in monkeys (16), rats (17), and goats (18), but the locations of these recordings have not correlated with specific GnRH perikarya (19). In fact, in the rat this multiunit electrical activity is most likely coming from GnRH axons or terminals, rather than from GnRH cell bodies (17). Recent data from an immortalized murine GnRH cell line (20) and from explants of monkey olfactory placode (21) suggest that pulsatile GnRH release is intrinsic to GnRH neurons. However, since both these approaches use embryonic GnRH neurons, or cells derived from them, it is questionable whether they are representative of the adult GnRH phenotype (22). It is also not clear what percentage of neurons contribute to the production of GnRH pulses in these in vitro preparations. Thus, these experimental approaches have failed to resolve whether a subset of GnRH neurons is responsible for either the LH surge or pulsatile LH secretion.

Another potential approach to addressing this issue is to monitor levels of Fos, or other immediate early gene (IEG) products. Stimulation-induced increases in IEGs have been observed in a wide variety of neural systems, and consequently the appearance of Fos has become accepted as a useful index of neuronal activation (23, 24). Fos has been used as a marker for activation of the GnRH neurons associated with the preovulatory LH surge in a number of species (25, 26, 27, 28, 29, 30). In general, Fos occurs in 50–60% of the GnRH perikarya at the time of the LH surge, but this expression is not limited to a particular anatomical subgroup. There are, however, two exceptions: in the hamster, the increase in Fos expression is only observed in more rostral GnRH perikarya (27) and, in the monkey, Fos is generally not found in GnRH neurons at the time of the surge (30). In this study, we have used Fos and FRA to determine whether a subgroup of GnRH neurons in sheep is activated in association with increased pulsatile LH secretion. Because these IEGs are often only expressed for a few hours after the initial activation (23, 24), we acutely stimulated episodic LH secretion using pharmacological and physiological approaches. Pharmacologically, we administered an antagonist to endogenous opioid peptides (EOPs) to luteal phase ewes. EOP antagonists increase pulsatile LH secretion in luteal phase ewes (31, 32) because EOPs mediate the negative feedback inhibition of GnRH and LH pulse frequency by progesterone (3). To physiologically stimulate episodic LH secretion, we exposed rams to ewes in behavioral estrus (33, 34).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Adult male and female sheep of mixed breeding were maintained in an open barn with free access to water and fed once daily with a maintenance regimen of silage. They were moved to indoor facilities 2–3 days before experimentation. Indoors, the animals were kept in individual pens under a photoperiod similar to that occurring outdoors. The routine handling of animals and experimental procedures involving animals were approved by the West Virginia University Animal Care and Use Committee.

Experimental protocols
Exp 1. This study was performed during the breeding season (October through January) in ewes that had demonstrated at least two normal 16- to 17-day estrous cycles (determined by monitoring estrous behavior with a vasectomized ram). Blood samples (3 ml) were collected by jugular venipuncture every 12 min for 4 h from ewes late in the luteal phase (days 9–12). At 2 h, nine of the ewes were injected iv with vehicle (5% dextrose) and the other six with 12.5 mg of WIN 44,441–3 (generously provided by Sterling-Winthrop, Rensselaer, NY). WIN 44,441–3 is an antagonist to EOP receptors that consistently increases pulsatile LH secretion in luteal phase ewes (31, 32). All animals were killed after the last blood samples and their heads were perfused with fixative. Serum was harvested and stored at -20 C until assayed.

Exp 2. This study was performed in July and August using 12 sexually experienced rams (2 vasectomized and 10 intact) that had been kept isolated from females for at least 6 weeks (33, 34). Estrous behavior was induced in ovariectomized ewes by sequential treatment with progesterone (20 mg given im on days 7, 5, and 3 before the experiment) and estradiol benzoate (100 µg im on the day before the experiment). On the morning of the experiment, ewes showing behavioral estrus were moved to the indoor facility. Blood samples were collected every 12 min for 4 h from males before exposure to females. The experimental rams (n = 7) were then moved into a pen in an adjacent room with 2 estrous females for every 2 or 3 males and sampling continued for an additional 2 h. Control rams (n = 5) were moved into another room and penned with 2 intact males for the final 2 h of blood collection. All rams were killed 2 h after exposure to ewes (or rams), and their heads were perfused with fixative. Serum was harvested and stored at -20 C until assayed.

Tissue preparation
After the last blood sample was collected, animals were heparinized (two iv injections of 25,000 U heparin given 10 min apart), then deeply anesthetized with sodium pentobarbital (~2,000 mg, iv), and rapidly decapitated. The heads were perfused via both internal carotids with 6 liters of 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.3) containing 0.1% sodium nitrite (a vasodilator) and 10 U/ml heparin. Tissue blocks containing the hypothalamus and rostral diencephalon were dissected out. The blocks extended from 1 cm anterior to the optic chiasm to the mamillary body in the rostral-caudal direction, were bounded by the hypothalamic fissures laterally, and extended approximately 0.5 cm dorsal to the anterior commissure. The tissue was stored in 4% paraformaldehyde at 4 C overnight and then placed in 30% sucrose at 4 C until infiltrated with sucrose. Thick (50 or 60 µm) frozen coronal sections were cut and stored at -20 C in a cryopreservative solution (35) for 1–3 months until being processed immunocytochemically for Fos/FRA and GnRH.

Assays
LH was measured in duplicate 200-µl aliquots in all samples using a previously described procedure (32) using anti-ovine LH (oLH) serum CSU204 (36). The average minimal detectable dose was 80 pg/tube (NIH-S24). The interassay and average intraassay coefficients of variation were 12.3 and 10.7%, respectively. Progesterone concentrations were measured in 100-µl aliquots of three samples from each ewe (32). The average minimum detectability was 35 pg/tube. The interassay and intraassay coefficients of variation were 15.8 and 9.2%, respectively.

Immunocytochemistry
Fos/FRA and GnRH were detected using a dual immunoperoxidase procedure that provides a permanent record of the location of single- and double-labeled cells (28). In this procedure, nuclear Fos/FRA was first detected using a modified avidin-biotin-immunoperoxidase procedure in which nickel-enhanced diaminobenzidine was used as the chromogen to produce a blue-black reaction product. The second antigen, GnRH, was then demonstrated using an avidin-biotin-immunoperoxidase procedure using diaminobenzidine without enhancement to produce a brown reaction product. For detection of Fos/FRA, a rabbit polyclonal antibody directed against a peptide corresponding to amino acids 128–152 (K-25, Santa Cruz Biotechnology, Inc., Santa Cruz, CA) was used at a dilution of 1: 40,000. This antibody also reacts with the Fos-related antigens (FRA-1, FRA-2) and Fos-B. For GnRH, we used a polyclonal rabbit antibody made against GnRH (LR-1 at 1:20,000, 48 h at 4 C, gift of R. Benoit).

Immunocytochemical controls included omission of one or both primary antibodies from the immunostaining protocol, and preabsorption of diluted antibody with purified peptide. Omission of either one of the primary antibodies in the double-label protocols eliminated immunostaining corresponding to that antibody and did not affect immunostaining corresponding to the other antibody. Preabsorption of either primary antibody with 0.1–1.0 µg/ml of the corresponding purified peptide (GnRH peptide obtained from Peninsula Laboratories, Inc. Belmont, CA; Fos/FRA peptide from Santa Cruz Biotechnology, Inc.) for 24 h at 4 C completely eliminated all subsequent immunostaining.

The number of single- and double-labeled GnRH-positive cells were counted in every fourth section (ewes) or every fifth section (rams) through the diencephelon. Cells were considered double labeled if a ring of GnRH-immunoreactive cytoplasm completely surrounded a Fos/FRA-positive nucleus, both viewed in the same plane of focus. For analysis, each coronal section was placed into one of four anterior-posterior regions, based on nuclei and major nerve tracks (15): the diagonal band of Broca (DBB), the POA, the anterior hypothalamic area (AHA), and the MBH. For each animal, the number and percentage of GnRH-immunoreactive cells in each region that contained Fos/FRA-positive nuclei were calculated. Fos-FRA positive cells were also counted in six sections through the MBH at levels extending from the rostral through the caudal median eminence. The lateral boundary of the MBH was defined by a parasagittal plane extending from the medial edge of the fornix to the ventral surface of the brain, and the dorsal boundary by the ventral edge of the fornix. All cell counts were performed by an observer who was blind to the identity of the individual animals.

Statistical analysis
LH pulses were differentiated from assay variability using three criteria previously described (32). Because LH pulse frequencies and the number and percentage of Fos-containing GnRH neurons were not normally distributed, differences between experimental and control groups in these variables were determined by the nonparametric Wilcoxon test. Differences in number of GnRH neurons in each hypothalamic area and in Fos/FRA-positive neurons between controls and experimental groups were determined using the unpaired t test. Statistical significance was set at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Exp 1
Based on progesterone measurements, all ewes were in the luteal phase and there was no difference in progesterone concentrations between WIN-treated (2.2 ± 0.2 ng/ml) and control (2.4 ± 0.3 ng/ml) ewes. In six of the vehicle injected controls, LH concentrations were undetectable in all serum samples; the other three controls each had one spontaneous LH pulse during the 4-h sampling period. The injection of WIN induced an LH pulse within a few minutes in five of the six ewes and significantly increased LH pulse frequency from 0.4 ± 0.3 to 1.2 ± 0.3 pulses/2 h in these ewes. Data from the ewe that failed to respond to WIN were omitted from subsequent analysis.

In the six control ewes that exhibited no LH pulses before death, very few GnRH neurons in the MBH and no other GnRH neurons expressed Fos/FRA (Figs. 1Go and 2Go, A and B), despite the presence of numerous Fos/FRA-positive cells in both the MBH (Fig. 2Go) and other hypothalamic areas. In contrast, in the WIN-treated ewes, approximately 30% of the MBH GnRH neurons contained Fos/FRA-positive neurons. There was a significant increase in both the percentage (Fig. 1Go and Fig. 2Go, C and D) and the number (Controls, 0.5 ± 0.3 vs. WIN, 8.6 ± 2.2) of Fos/FRA-containing GnRH neurons in the MBH of the WIN-treated animals. However, GnRH cells in the POA and elsewhere did not contain Fos/FRA-positive neurons (Fig. 1Go). Interestingly, in the three control ewes that had endogenous LH pulses, the percentage (46.1 ± 21.9%) and the number (8.3 ± 1.7) of Fos/FRA-positive GnRH cells in the MBH were similar to those in the WIN-treated animals; GnRH perikarya in other areas from these ewes did not express Fos-FRA in their nuclei.



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  		Figure 1. The mean (± SEM) number of GnRH neurons (top panel) and percentage of GnRH neurons coexpressing Fos/FRA (bottom panel) in different regions of the diencephalon of five ewes treated with WIN (hatched bars) and of six control ewes that had no LH secretion in the 4 h before tissue collection (open bars). *, P < 0.05 vs. controls.

 


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  		Figure 2. Dual immunoperoxidase demonstration of nuclear Fos/FRA and cytoplasmic GnRH in comparable coronal sections through the MBH of a control ewe (A and B) and a WIN-treated ewe (C and D). GnRH cells indicated by the open arrows in A and C are shown at higher magnification in B and D, respectively. Very few of the GnRH cells located in the MBH of the control ewe expressed Fos/FRA despite the presence of Fos/FRA-positive cells in adjacent tissue, whereas there is clear Fos/FRA expression in the GnRH neuron in the WIN-treated animal. Solid arrows indicate GnRH terminals in the rostral median eminence. IIIv, Third ventricle. Bar in C = 100 µm; bar in D = 20 µm.

 
The distribution of GnRH neurons throughout the diencephalon was similar in the two groups, with most GnRH neurons being in the POA and AHA (Fig. 1Go). The number of GnRH neurons observed in the MBH was quite variable (range of 7–109), but was not affected by treatment (Fig. 1Go) or the presence (36.6 ± 11.7, n = 8) or absence (35.1 ± 11.4, n = 7) of LH pulses before death. Similarly, there was considerable variability among ewes in Fos/FRA-positive nuclei (Fig. 2Go), but WIN treatment did not affect their overall number in representative sections of the MBH (129 ± 43 for WIN-treated vs. 115 ± 37 for controls).

Exp 2
LH pulse frequency during the first 2 h of this experiment averaged 0.8 pulses/2 h and did not vary between control and experimental animals. In the five control rams exposed to another male, either a single LH pulse (three rams) or no pulses (two rams) occurred in the last 2 h of sampling so that pulse frequency did not change (Fig. 3Go). In contrast, six of the seven males exposed to estrous females had two or three pulses during the last 2 h, and LH pulse frequency increased approximately 2-fold in this group (Fig. 3Go). Data from the ram that failed to respond to estrous females were not included in subsequent analysis.



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  		Figure 3. Effect of introduction of estrous ewes (bottom panels) or rams (top panels) on pulsatile LH secretion in male sheep. The left panels depict representative LH pulse patterns in two rams from each group. The bars on the right depict mean (± SEM) LH pulse frequency before (open bars) and after introduction of females or males. *, P < 0.05 vs. pretreatment value.

 
In all six of the rams that responded to estrous females, Fos/FRA-positive GnRH neurons were observed, whereas this colocalization was only observed in one of five control rams (P < 0.05, {chi}2 test). The expression of Fos/FRA was largely limited to MBH GnRH neurons (Fig. 4Go). No GnRH neurons in the DBB or the AHA showed Fos/FRA expression. In two of the experimental, and one of the control, rams a few (1, 2) POA GnRH neurons expressed Fos/FRA, but this response was not affected by treatment. In contrast, there was more than a 10-fold increase in the percentage (Fig. 4Go) and number (7.8 ± 3.0 vs. 0.2 ± 0.2, P < 0.01) of MBH GnRH neurons expressing Fos/FRA in the rams exposed to estrous females.



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  		Figure 4. The mean (± SEM) number of GnRH neurons (top panel) and percentage of GnRH neurons coexpressing Fos/FRA (bottom panel) in different regions of the diencephalon of six rams exposed to estrous females (hatched bars) and five controls exposed to other males (open bars) 2 h before tissue collection. *,P < 0.05 vs. controls exposed to males.

 
There was no difference between groups in either the distribution of GnRH neurons (Fig. 4Go) or the number of Fos/FRA-positive nuclei in representative sections of the MBH (treated, 404 ± 18; controls, 440 ± 24). There was considerable variability among rams in the total number of GnRH neurons identified (range 143–717), and this was also evident within the MBH (range 21–201).

To compare the results obtained in Exp. 1 and Exp. 2, we calculated the total number of GnRH neurons within the MBH that contained Fos/FRA in each sheep. As illustrated in Fig. 5Go, when these data were plotted against the increase in LH pulse frequency during the last 2 h before death there was a strong positive regression (R = 0.76; P < 0.001 by linear regression). The total number of Fos/FRA-positive MBH GnRH neurons was similar in ewes (42 ± 1.4; n = 7) and rams (33.6 ± 14.0; n = 7) that had an increase of one or more LH pulses during this period, and these numbers were significantly greater than those in ewes (3.5 ± 2.2; n = 8) and rams (2.0 ± 2.0; n = 5) that failed to show such an increase.



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  		Figure 5. The correlation between Fos/FRA expression in GnRH neurons in the ovine MBH and the increase in LH pulse frequency in the 2 h before tissue collection. For each animal the total number of GnRH neurons expressing Fos/FRA was calculated by correcting the observed number of GnRH neurons containing Fos/FRA for the sampling frequency (every fourth section for ewes; every fifth section for rams). The increase in LH pulse frequency was calculated by subtracting the mean frequency (pulses/2 h) before treatment from the LH pulse frequency during the last 2 h. Data from ewes are depicted by circles; data from rams by triangles. Data from controls are depicted by open symbols; data from treated animals by closed symbols. Regressional analysis resulted in P < 0.001.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
These data demonstrate that both a pharmacological and a physiological stimulus that increased pulsatile LH release also induced Fos/FRA expression in GnRH neurons, but only in those GnRH neurons found in the MBH. They are thus consistent with the hypothesis that stimulation of pulsatile GnRH secretion by these agents involves a subset of GnRH neurons located in the MBH. This interpretation is based on the assumption that Fos/FRA expression is a useful index of neuronal activation. While neural systems can be activated without concomitant Fos/FRA expression (37), the association of the two is sufficiently common that Fos/FRA expression is now generally accepted as an indication of an increase in neural activity (23, 24). Thus, it has been useful in mapping the GnRH neurons contributing to the preovulatory LH surge in a number of species (25, 26, 27, 28, 29, 30). Even if, in the case of these experiments, there was increased activity of GnRH neurons outside the MBH without Fos/FRA expression, then the mechanisms responsible for stimulation of these GnRH neurons must be different from those stimulating the GnRH neurons in the MBH. The results would thus still support the hypothesis that MBH GnRH perikarya represent a discrete subset of neurons, which are controlled by different neuroendocrine mechanisms than GnRH perikarya located elsewhere in the ovine diencephalon. Similar results have recently been reported in the rat, where naloxone has been shown to increase the percentage of GnRH cells expressing Fos in the lateral hypothalamic area, the most caudal portion of their distribution (38). In that study, however, naloxone also induced Fos in some POA GnRH neurons. In fact, more POA, than MBH, GnRH neurons expressed Fos after naloxone, although the percentage of POA GnRH neurons expressing Fos was much lower because of the higher number of GnRH neurons in the POA.

One potential explanation for the selective activation of MBH GnRH neurons with both stimuli is that these perikarya are preferentially inhibited by EOPs. Since EOPs may mediate some of the negative feedback action of testosterone in rams (39), the increase in LH pulse frequency in rams could reflect a decrease in EOP tone that would mimic the effect of the EOP antagonist in ewes. The hypothesis that EOPs inhibit MBH GnRH neurons is supported by previous work demonstrating that local administration of EOP antagonists within the MBH (40, 41) increased pulsatile LH secretion in luteal phase ewes. However, these antagonists were also effective when placed in the POA of luteal phase ewes (40, 41). There is no direct evidence indicating where EOPs act in the ram to inhibit GnRH pulse frequency. It is also not clear whether EOPs act directly on GnRH neurons in the sheep. There is evidence for EOP-GnRH synapses in the MBH of the monkey (42) and rat (43); whether such synapses also occur in sheep remains to be determined.

The limitation of Fos/FRA to MBH GnRH neurons when episodic LH secretion increases is in marked contrast to Fos expression associated with induction of the preovulatory LH surge. At the time of the LH surge in the ewe, Fos is observed in approximately 40–50% of GnRH neurons regardless of whether the GnRH perikarya are located in the DBB, POA, AHA, or MBH (28). A similar distribution is observed with an antiserum that detects only Fos (28) and the antiserum used in this study that detects both Fos and FRA (44). This difference raises the possibility that a subset of GnRH neurons within the MBH of the sheep is responsible for episodic GnRH and, hence, LH secretion in this species. This possibility is apparently not consistent with the ability of anterior deafferentation to decrease LH pulse amplitude (13). There are, however, several possible explanations for this apparent discrepancy. First, deafferentation could disrupt stimulatory neural input to the GnRH neurons in the MBH. Second, deafferentation might disrupt projections from MBH GnRH neurons to GnRH neurons in more rostral areas. Finally, MBH GnRH neurons might innervate GnRH terminals in the median eminence that arise from rostral GnRH perikarya; if so, deafferentation would eliminate these terminals, thus decreasing the amount of GnRH released per pulse. The latter possibility is consistent with the occurrence of GnRH-GnRH synaptic contacts in the ovine median eminence (45).

Previous attempts to correlate Fos expression and episodic LH secretion have been largely unsuccessful. In the sheep, there is no difference in the number of GnRH neurons containing Fos between luteal phase and ovariectomized ewes (28). However, in this study, tissue was collected from ovariectomized ewes 2 days after progesterone withdrawal so that a transitory increase in Fos expression would have been missed. It is interesting to note that the GnRH neurons that contained Fos were all found in the MBH (28). In the rat and mouse (25, 26), little Fos expression is observed in GnRH cells at times other than the LH surge, even though pulsatile LH secretion occurs at these times. In the reflex-ovulating ferret, Fos expression occurs in 15–20% of the GnRH neurons in unmated females and males, but this expression is not anatomically localized (29). On the other hand, in the hamster GnRH neurons in the caudal POA expressed Fos on all days of the estrous cycle, suggesting that this subset of GnRH neurons may be involved in episodic GnRH release (27). One possible explanation for these differences is that the work reported here and in the hamster used an antibody that detected both Fos and FRA, while the other work was done with antisera that detected only Fos.

We observed a large amount of variability across animals in both the total number of GnRH neurons and the number of GnRH neurons within the MBH. This variability is consistent with previous reports in the sheep, when comparisons are made across and within studies (15, 46, 47, 48). One possible explanation for these observations is that there may be considerable redundancy within the GnRH system. For example, there is strong evidence that much more GnRH is released during the preovulatory GnRH surge than is needed to induce the preovulatory LH surge (49). Such data suggest that the normal complement of GnRH neurons is more than sufficient to maintain fertility. If so there would be little evolutionary pressure to select a specific number of GnRH neurons, and one would predict considerable variability among animals.

If GnRH neurons in the ovine MBH are responsible for episodic GnRH secretion, then a maximum of approximately 100 perikarya must be sufficient because this is the approximate number of GnRH neurons in this area of the ovine diencephalon (15, 46, 47, 48). Thus, these results raise the interesting question: how many GnRH neurons are needed to produce a GnRH pulse? While there is no definitive answer to this question, there are data consistent with the proposal that a small percentage of the total population may be involved. In the sheep, if one compares the amount of GnRH released during the surge (~4,000 pg, calculated from data presented in Refs. 3, 5) with that released during a pulse [20–40 pg, (5)], one would conclude that 1% of the GnRH perikarya are sufficient. While this is an overly simplistic comparison because of the different hormonal milieu required for pulses and surges and the different time frames over which secretion occurs, data from rodents also support the concept that a small number of GnRH neurons are sufficient to maintain pulsatile LH release. In rats, frontal hypothalamic deafferentation at the posterior border of the optic chiasm blocks the preovulatory LH surge, but does not produce any decrease in pulsatile LH secretion (7, 8). This continuation of episodic LH secretion has been attributed to a subchiasmatic projection from the POA to the median eminence (9). Since this pathway represents a very small percentage of the total projections from the POA to the MBH in the rat (9), this explanation implies that a small percentage of GnRH perikarya are sufficient to maintain pulsatile LH secretion. Finally, there is direct evidence from studies in which GnRH neurons were transplanted into hypogonadal mice that 1–25 GnRH neurons are sufficient to maintain LH pulses in this species (50).

In conclusion, the results of this study suggest that a subset of GnRH neurons located in the ovine MBH are responsible for the increase in episodic LH secretion induced by an EOP antagonist in ewes and by exposure of rams to estrous females. They also raise the possibility that these neurons may be responsible for episodic GnRH secretion in the sheep.


   Acknowledgments
 
We thank Ms. Shelley Jones for technical assistance and Dr. Gordon Niswender, Dr. Leo Reichert, Jr., and the National Pituitary Agency for RIA reagents.


   Footnotes
 
1 Funded by NIH Grant HD-17864 and United States Department of Agriculture Grant 9702249. Back

2 Present address: Départment de Reproduction Animale et d’I. A, Institut Agronomique et Vétérinaire Hassan II, BP 6516-Instituts, 10101-Rabat, Morocco. Back

3 Present address: Department of Biomedical and Health Sciences, Grand Valley State University, Allendale, Michigan 49401. Back

Received June 30, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Everett JW, Sawyer CH, Markee JE 1949 A neurogenic timing factor in control of the ovulatory discharge of luteinizing hormone in the cyclic rat. Endocrinology 44:234–250[Medline]
  2. Halasz B, Gorski RA 1967 Gonadotropic hormone secretion in female rats after partial or total interruption of neural afferents to the medial basal hypothalamus. Endocrinology 80:608–622[Abstract/Free Full Text]
  3. Goodman RL 1994 Neuroendocrine control of the ovine estrous cycle. In: Knobil E, Neill JD (eds) The Physiology of Reproduction, ed 2. Raven Press Ltd, New York, vol 2:659–710
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