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Endocrinology Vol. 138, No. 10 4220-4226
Copyright © 1997 by The Endocrine Society

ARTICLES

Does a Short Loop Feedback Mechanism for the Control of Luteinizing Hormone Secretion Exist in the Ewe?1

Donal C. Skinner2, Neil P. Evans and BenoÎt Malpaux

Laboratory of Neuroendocrinology, The Babraham Institute (D.C.S., N.P.E.), Babraham, Cambridge, United Kingdom CB2 4AT; and Institut National de la Recherche Agronomique, Unité de Neuroendocrinologie Sexuelle, Station de Physiologie de la Reproduction (D.C.S., B.M.), Nouzilly 37380, France

Address all correspondence and requests for reprints to: Dr. Donal C. Skinner, Station de Physiologie de la Reproduction, Nouzilly 37380, France. E-mail: skinner{at}tours.inra.fr


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
It is not known whether a short loop feedback mechanism for the regulation of LH exists in sheep. This study on ovariectomized ewes investigated whether a bolus injection (10, 1, and 0.1 µg LH or 1 µg BSA; n = 4) or a 3-h continuous infusion of exogenous LH (100 or 1 ng/min; n = 7) into the third ventricle through a permanent indwelling cannula could influence the activity of the GnRH pulse generator, as determined by measurement of endogenous LH secretion. To assess the potential for involvement in a LH short loop feedback system and to estimate the level of LH in the hypothalamic milieu, the concentrations of LH in the peripheral circulation, portal circulation, and third ventricle were measured during an estradiol-induced preovulatory LH surge (n = 4).

Neither the bolus nor continuous administration of LH into the third ventricle had any effect on the mean interpulse interval, nadir, pulse amplitude, or circulating level of systemic LH. Furthermore, despite portal LH concentrations being more than 20-fold higher than jugular LH concentrations, LH levels in third ventricular cerebrospinal fluid remained barely detectable and did not reflect dynamic secretory events in the peripheral or hypothalamo-hypophyseal portal blood. These data demonstrate that in ewes, little pituitary LH reaches the third ventricle, and the small amount that does is unable to affect peripheral gonadotropin release. Our study suggests, therefore, that a short loop feedback system for LH does not exist in the ewe.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE PRESENCE of a short loop feedback mechanism for the regulation of LH secretion in mammals (1), whereby LH acts back on the hypothalamus to modulate GnRH release, has long been a controversial topic. Interest in such a physiologically active short loop system has recently been rekindled by suggestions that it may be an essential pathway by which melatonin modulates reproductive activity (2, 3) and that it may contribute to the inhibition of LH secretion after the preovulatory LH surge (4).

Studies in the rat and pig have provided experimental evidence to support the existence of a short loop feedback mechanism for LH (5, 6). Moreover, specific LH receptors have recently been detected in a number of brain areas in the cow, rat, and human (7). Indeed, in the rat, LH receptors were detected in the preoptic area, which in both this species (8) and the sheep (9, 10) is the location of the majority of GnRH neuronal perikarya. Thus, it would appear that, mechanistically, LH could act within the brain to modify the release of GnRH. Studies in other species, however, have not provided evidence to support this proposal. In the primate, no evidence has been found to support the existence of a short loop regulatory system (11, 12, 13, 14, 15), and studies in sheep have been inconclusive. In the sheep, neither Coppings and Malven (16) nor Caraty and co-workers (17) were able to document the existence of a LH regulatory short loop feedback system after manipulation of circulating gonadotropin levels. However, Padmanabhan and colleagues (18) recently noted an increase in GnRH secretion after administration of a GnRH antagonist. This result could be explained in two ways: by the existence of an ultrashort loop feedback system for GnRH or by a short loop feedback system for LH, with the reduction in LH concentrations after antagonist administration stimulating an increase in GnRH release. It was not possible to discern which system may have been operative in their study, but these researchers strongly favored the ultrashort loop hypothesis and suggested that the discrepancy between the two studies was due to a masking of the effect by a high endogenous pulse frequency in the study of Caraty et al. (17).

Alternatively, if the results of Padmanabhan et al. (18) were due to a short loop feedback system, then the apparent discrepancy may lie with the source and concentration of LH seen by the GnRH neurons and the size of the LH changes induced by the different experimental paradigms. In this regard, as neural LH probably originates from a portal and/or pars tuberalis source, and data exist to suggest that portal LH concentrations may be 50- to 100-fold higher than jugular LH levels (19), it is possible that peripheral LH manipulations, such as those used by Coppings and Malven (16), had little effect on this median eminence-apposed pool. Similarly, the 2-fold increase in the peripheral LH concentration induced by GnRH agonist treatment in the study by Caraty et al. (17) may also have had little or no effect on the concentration of LH in the median eminence. Thus, if the results obtained by Padmanabhan et al. (18) were due to a short loop feedback system, it could be argued that the changes in GnRH secretion were induced by the reduction in LH secretion caused by the GnRH antagonist and that their treatment had a more physiological effect on the concentration of LH seen by the GnRH neurons.

In sheep, the GnRH perikarya are located predominantly in the organum vasculosum of the lamina terminalis/preoptic area, with their terminals in the median eminence. The GnRH system, therefore, is closely associated with the third ventricle (9, 10). Studies in rats have demonstrated that after intracerebroventricular (icv) administration, peptides as large as horseradish peroxidase readily penetrate the tissue that surrounds the cerebral ventricles (20). Furthermore, due to the association of the cerebrospinal fluid (CSF) with the extracellular fluid of the brain, hormone levels in the CSF probably reflect the concentrations to which neural tissues are exposed (21). Thus, it is possible that the administration of LH via the ventricular system and measurement of LH in ventricular CSF may provide a more reliable method of evaluating the existence of a short loop feedback system than manipulation of peripheral gonadotropin concentrations (16, 17, 18).

The objectives of the present study, therefore, were 2-fold: 1) to determine whether exogenous LH, administered into the third ventricle of the ewe, could influence the activity of the GnRH pulse generator, as estimated from endogenous LH levels in the peripheral circulation; and 2) to determine the precise relationship between the concentrations of LH in portal and jugular blood and third ventricular CSF to allow consideration of the physiological importance of this route for the regulation of GnRH secretion.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Studies were conducted at either the INRA Unité de Neuroendocrinologie Sexuelle (Nouzilly, France) or at The Babraham Institute (Cambridge, UK), but the maintenance and preparation of ewes at each site were similar. Specifically, ewes were ovariectomized at least 1 month before experimentation, were housed thereafter in rooms with natural photoperiod, had free access to water, and were fed daily with hay, straw, and corn. In Exp 1 (part 2), 2, and 3, ewes were restrained so that they could not turn around, but were able to lie down and move forward and backward freely. To prevent the stress of social isolation, ewes were always maintained with other sheep.

Surgery
Guide cannulas (17 gauge, 42 mm, stainless steel luer-lock needle; Coopers Needle Works, Birmingham, UK) were introduced stereotaxically into the third ventricle using a modification of a method described previously (22). Briefly, under halothane anesthesia, the head of the ewe was positioned in a stereotaxic frame, and 1 ml radioopaque liquid (Omnipaque, Nycomed Ingenon, France; or Ultravist, Schering, Germany) was injected into the right lateral ventricle. Using the specific landmarks of the ventricular system, the cannula tip was positioned 1 mm in front of the vertical and 1 mm below the horizontal, tangents of the intermediate mass. Correct placement was ascertained by the free flow of CSF back up the cannula. The cannula was plugged and fixed in place with acrylic dental cement, and a stainless steel or Teflon cap was placed around it for protection. At the time of the experiment, a polyethylene or SILASTIC brand catheter (Dow Corning, Midland, MI) was inserted through the guide cannula so that the distal tip ended at the tip of the guide cannula.

After establishing that CSF still flowed up the ventricular guide cannula, the portal blood collection apparatus was implanted at least 2 weeks before an experiment using a procedure described in detail previously (23). At the time of the experiment, ewes were heparinized, and portal blood was collected into glass cylinders containing 3 ml 3 x 10-3 M bacitracin. After portal blood collection, ewes were killed by an overdose of sodium pentobarbitone (Lethobarb, Duphar Veterinary, UK), and the pituitary glands were inspected to verify the site of lesion.

Exp 1: effect of bolus icv LH injections on LH secretion
Part 1.
The aim of this study was to determine whether central administration of LH (1051-CY-LH) would affect LH secretion, as measured in the peripheral circulation. Four Ile-de-France ewes (Nouzilly, France; March) bearing icv guide cannulas were treated with three amounts of LH (10, 1, and 0.1 µg) or 1 µg BSA (Sigma Chemical Co., St. Louis, MO), administered on separate consecutive days in a random order. Jugular blood samples were collected by venepuncture every 10 min for 6 h, 3 h before and 3 h after initial icv LH administration. All blood samples were centrifuged (20 min, 1500 x g, 4 C), and the plasma stored at -20 C until assayed for LH. LH and BSA for icv administration were dissolved in Ringer’s lactate (Bruneau, Paris, France) and given as a 50-µl injection over 30 sec via a polyethylene catheter. BSA and the 1- and 0.1-µg doses of LH were injected three times: 3, 4, and 5 h after the onset of sample collection. As we did not know how much icv LH would enter the peripheral circulation, and if it did we did not want to obscure endogenous LH secretion, the 10-µg LH dose was injected only once, at 3 h.

Part 2.
It is possible that the LH injected into the third ventricle in the previous experiment was rapidly removed from the CSF by enzymatic action or some binding protein. To address this caveat, integrated CSF samples were collected (30 µl/min) from four Ile-de-France ewes (Nouzilly, France; April) for 5 h after a single bolus icv injection of 10 µg LH. Three 15-min samples were collected immediately before the injection to determine endogenous CSF-LH concentrations.

Exp 2: effect of continuous icv LH infusion on LH secretion
The effect of a constant icv infusion of exogenous LH on LH secretion into the peripheral circulation was investigated in adult Welsh mountain ewes (n = 7; Babraham, UK; October). A constant rate infusion pump (Syringe Driver Type MS 16A, Graseby Medical, Watford, UK) was connected to each icv cannula and, to allow any possible perturbations caused by the introduction of the catheter to dissipate, artificial goat CSF (ACSF; NaCl, 7.4 g/liter; NaHCO3 1.9 g/liter; NaH2PO4·2H2O, 15.8 mg/liter; Na2HPO4·2H2O, 71.5 mg/liter; KCl, 223 mg/liter; MgSO4, 120 mg/liter; CaCl2, 128 mg/liter) (24) was infused for 3 h before samples were collected. Jugular blood samples (10 min) were collected by venepuncture for the following 9 h, and the plasma was removed and stored at -20 C for LH analysis. For the first and last 3 h of the experiment, ACSF alone was infused. Using a cross-over design, either 100 ng/min (18 µg/3 h) or 1 ng/min (180 ng/3 h) LH (NIH LH-S18) was infused from 3–6 h. The experiment was repeated 4 days later when ewes were infused with LH at the alternate rate.

Exp 3: comparison of LH in CSF, portal blood, and jugular blood during the preovulatory surge
To determine the concentration of LH in portal blood, how this compared with LH concentrations in the jugular vein, and whether LH was present in ovine CSF, seven ewes that had been implanted with third ventricular CSF-withdrawal guide cannulas were prepared for portal blood collection (Babraham, UK; May–November). Due to technical problems, hypothalamo-pituitary portal blood and CSF could only be simultaneously collected from four animals.

A LH surge was induced in all ewes using the model of Goodman et al. (25). Briefly, a 10-mm SILASTIC capsule containing crystalline 17ß-estradiol was inserted sc, and two progesterone implants (CIDR, InterAg, Hamilton, New Zealand) were placed intravaginally. After 10 days, progesterone was removed, and 24 h later, four 30-mm SILASTIC estradiol capsules were implanted sc. These estradiol capsules produce a circulating estradiol concentration of approximately 7 pg/ml, which is comparable to the amount circulating during the peak follicular phase (25, 26). Hourly instantaneous jugular and integrated portal blood samples were collected for measurement of LH concentrations from 12–48 h after estradiol insertion. In a pilot study, we had found that LH concentrations in unextracted CSF were below the detection limit of the assay (data not shown), and thus, CSF-LH was estimated in extracted samples according to the following method. Hourly samples of CSF (1.8 ml/h) were collected directly into methanol (3 ml) and centrifuged (30 min, 1500 x g, 4 C). The supernatant was discarded, and the precipitate was dried and stored (-20 C) until assayed for LH. At the time of assay, the pellet was resuspended in assay buffer (250 µl). Using this method, recovery of LH (NIH-S11) added to ACSF samples exceeded 90%.

RIA
Plasma and CSF samples were assayed for LH in duplicate 100-µl aliquots using a previously described method (27, 28) (Nouzilly assay for Exp 1) or the RIA method of Niswender et al. (29) (Babraham assay for Exp 2 and 3). All samples from an individual ewe were run in the same assay. The inter- and intraassay coefficients of variation of the Nouzilly assay (two assays) averaged 8% and 11%, respectively, and assay sensitivity was 0.1 ng/ml standard 1051-CY-LH (i.e. 0.2 ng/ml NIH-S1). The inter- and intraassay coefficients of variation of the Babraham assay averaged 7% and 13%, respectively, and assay sensitivity was 0.2 ng/ml standard NIH-S11 (four assays).

Analysis
LH data from Exp 1 and 2 were analyzed as described previously (21) using the Munro algorithm (30), which is a modified version of the Pulsar algorithm (31). The mean interpulse interval, nadir, pulse amplitude, and circulating level of LH for each ewe were calculated by the program.

In Exp 1, data were analyzed statistically by a two-way repeated measures ANOVA [period (preinjection/postinjection) and dose]. Data from Exp 2 were analyzed using a Latin square, repeated measures ANOVA design [period (preinfusion/infusion/postinfusion) and dose]. Statistical analyses were made using SuperANOVA (Abacus Concepts, Berkeley, CA).

In Exp 3, the onset of the jugular, portal, and CSF LH surges were defined as the first sample in these compartments that exceeded the presurge baseline by 2 SD of this baseline and did not return to baseline within 2 h. The presurge baseline and SD for each compartment were calculated from the samples collected for the first 4 h of the experiment. The amplitude of the surge was taken as the peak level after insertion of the estradiol minus the presurge baseline. Statistical comparisons between jugular plasma and portal plasma LH concentrations were made with Student’s t test for paired data. All data are presented as the mean ± SEM.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Exp 1: effect of bolus icv LH injections on LH secretion
Part 1.
LH profiles from a representative ewe (no. 17) are illustrated in Fig. 1Go, and mean LH secretory characteristics shown in Table 1Go. The icv injection of BSA had no effect on the mean LH pulse amplitude, interpulse interval, nadir, or circulating level. Similarly, no effect on any parameter of LH release was seen in response to the 10-, 1-, or 0.1-µg LH challenge.



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  		Figure 1. A representative result (ewe 17) illustrating the effect of the 10-, 1-, and 0.1-µg LH and 10-µg BSA injections on pituitary LH release into the peripheral circulation. Arrows indicate times of injection, which were administered over 30 sec. Detected pulses are denoted by solid symbols.

 

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  		Table 1. Effect of bolus icv LH injections on LH secretion

 
Part 2.
The change in the LH concentration in the third ventricle after a 10-µg bolus injection is shown in Fig. 2Go. LH was undetectable in preinjection CSF samples. Fifteen minutes after the injection, the LH concentration averaged 107.2 ± 13.9 ng/ml, whereafter levels fell at a constant rate until the end of the experiment, when the LH concentration averaged 3.7 ± 1.8 ng/ml.



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  		Figure 2. The mean (±SEM) concentration of LH in the third ventricle after a 10-µg bolus injection (arrow). CSF samples (30 µl/min) were collected for 5 h after the injection, and LH was still detectable in the CSF at the end of the experiment.

 
Exp 2: effect of continuous icv LH infusion on LH secretion
Figure 3Go illustrates representative results from an ewe (no. 2W46) infused with LH into the third ventricle at rates of 100 and 1 ng/min. Neither rate of LH infusion had any effect on any parameter of endogenous LH secretion (Table 2Go).



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  		Figure 3. A representative result (ewe 2W46) illustrating the effect of the 100 and 1 ng/min LH infusions on pituitary LH release into the peripheral circulation. Shaded bars indicate the period of LH infusion; ACSF was infused for the first and last 3 h of the experiment. Detected pulses are denoted by solid symbols.

 

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  		Table 2. Effect of continuous icv LH infusion on LH secretion

 
Exp 3: comparison of LH in CSF, portal blood, and jugular during the preovulatory surge
In response to the stimulation by estradiol, a LH surge was clearly observed in the peripheral blood of all ewes. The LH surge was also evident in portal blood, but no significant increase in LH concentrations was seen in the CSF. After extraction and reconstitution in a minimal amount of assay buffer, LH concentrations in CSF remained extremely low and exceeded the detection limit of the assay in three of the four ewes (Fig. 4Go). In contrast, portal LH concentrations were very high (mean basal, 63.9 ± 23.4 ng/ml; mean surge, 204.8 ± 50.4 ng/ml; mean maximum, 458.9 ± 119.6 ng/ml) and exceeded jugular LH levels (mean basal, 2.0 ± 0.8 ng/ml; mean surge, 9.4 ± 0.8 ng/ml; mean maximum, 22.8 ± 4.1 ng/ml). The concentration of LH in portal blood was 22 ± 4-fold higher than the concentration in jugular blood calculated over the whole collection period. The onsets of the LH surge in portal blood (17.1 ± 0.9 h after estradiol insertion) and jugular blood (17.8 ± 1.0 h) and the durations (portal, 12.3 ± 2.4 h; jugular, 13.0 ± 2.9 h) of the surges were similar.



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  		Figure 4. The portal, jugular, and CSF concentrations of LH in ewe 426. LH was extracted from the CSF, and values shown are corrected for the CSF sample volume. The detection threshold for the extracted LH was 0.018 ng/ml. In all ewes (n = 4), the LH surge occurred simultaneously in portal and jugular compartments, and portal LH concentrations were significantly (P < 0.01) greater than jugular LH concentrations.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study suggests strongly that a short loop feedback mechanism for LH does not exist in the ovariectomized ewe. Neither the mean LH pulse amplitude, pulse frequency, nadir, or concentration was affected by either a bolus or a continuous infusion of LH into the third ventricle.

The existence of a short loop feedback mechanism for LH has long been hypothesized, but experimental evidence across different species varies. Kawakami and Sawyer (32) first suggested that LH may act via a short loop feedback mechanism on account of the effect of exogenous LH on electroencephalographic recordings. Subsequent studies in rats reported that ovarian activity was significantly inhibited by long term LH microimplants positioned directly in the median eminence (33) and that these implants suppressed circulating LH and GnRH concentrations (34, 35). More recently, it has been reported that short term administration of LH into the third ventricle of the rat is capable of suppressing endogenous LH and that this effect occurs through a neural, and not a pituitary, site of action (6). This finding has been supported by studies on median eminence/pars tuberalis explants, which were found to release more GnRH when exposed to LH antiserum, suggesting that endogenous LH inhibited terminal GnRH release (2). The possible existence of a short loop mechanism for the regulation of LH secretion has also been observed in the pig, in which iv administration of hCG inhibited the LH surge (5).

The mechanisms by which LH could act to attenuate GnRH release are poorly understood, but in rats may involve modulation of arcuate nucleus/median eminence neuronal (36, 37, 38) or enzyme (39) activity. The recent discovery of LH receptor messenger RNA in the hypothalamus has added credence to this hypothesis (7). It is also noteworthy that Mores and colleagues (4) recently reported that LH inhibits GnRH release from immortalized GnRH (GT1–7) neurons. These neurons are know to express LH receptors (40), but the physiological bearing of these results remains to be established.

The results from the present study strengthen findings obtained from earlier investigations in sheep that were unable to demonstrate the presence of a short loop feedback mechanism in this species. Those studies manipulated peripheral LH concentrations by either applying a GnRH antagonist (Nal-Glu, which decreased circulating LH levels) and agonist (D-Trp6 GnRH, which increased LH concentrations) (17) or administering exogenous LH (16). These data also provide insight into results obtained by Padmanabhan and colleagues (18), who recently reported that Nal-Glu caused an unexpected increase in portal GnRH levels. In discussion of their results, the researchers concluded that the data could be explained by the existence of either a short or an ultrashort loop feedback mechanism, but that from the data obtained it was not possible to rule out either possibility. The present study supports their suggestion that their results were unlikely to be caused by a LH short loop feedback mechanism. Whether a physiological ultrashort loop mechanism exists in sheep remains equivocal (17, 18, 41).

Studies in monkeys and humans have also failed to demonstrate an operative short or ultrashort loop feedback system for LH (11, 12, 13, 14, 15). Specifically, Knobil (11) found that endogenous gonadotropin release in ovariectomized rhesus monkeys was unaffected by peripheral administration of exogenous noncross-reacting, but biologically active, gonadotropins. Furthermore, the GnRH agonist buserilin, which caused a 2-fold increase in peripheral LH concentrations, did not influence electrophysiological recordings of hypothalamic neural activity associated with GnRH release in this species (12). Knobil and colleagues recently substantiated these earlier findings by demonstrating that neither icv GnRH, which significantly increased peripheral LH concentrations, nor icv antide, a GnRH antagonist that suppressed circulating LH levels, affected the GnRH pulse generator (15). Similarly, in humans, peripheral hCG administration had no effect on endogenous preovulatory LH surges despite its ability to bind to the LH receptor (14).

The extremely high concentrations of LH noted in portal blood in our study and others (19, 42) are of significant interest with regard to the possible existence of a short loop feedback system for the regulation of LH secretion. If portal LH levels measured in this study reflect true in vivo portal concentrations, this suggests that despite a close association between the portal vessels and the median eminence, extremely little LH is able to cross the short distance from the portal vessels to the third ventricle. Thus, little portal LH probably penetrates neural tissue in vivo and is, therefore, unlikely to influence neuronal activity within the median eminence area. It has also been suggested recently that a short loop regulatory feedback system for LH may contribute to the termination of the preovulatory LH surge (4). If this was a contributing factor to the termination of the surge, then we could expect the GnRH surge to terminate either before or coincidentally with the portal and jugular LH surges. As both portal GnRH (43) and CSF GnRH (22) surges outlast the LH surge by several hours, it is clear that a short loop mechanism cannot be operating to influence GnRH release at this time because GnRH concentrations are still elevated while LH is at a basal level.

The source of the high LH concentration in the hypophyseal portal blood is also worth comment. The long portal vessels penetrate the pars distalis after first passing through an area of the pituitary gland called the zona tuberalis, a ventral extension of the pars tuberalis (44, 45). Blood entering the pars distalis, therefore, has already been exposed to the environment of the pars/zona tuberalis. In portal blood studies in sheep, portal vessels are lesioned in the zona tuberalis (23). This zona tuberalis area has comparatively more gonadotropes than surrounding adenohypophyseal tissue in ewes (45). Thus, LH in portal blood could originate from the pars/zona tuberalis region, which would suggest that there may be two sources of this gonadotropin: one originating from the pars/zona tuberalis complex, which is secreted into portal vessels and passes through the pars distalis before entering the systemic circulation, and the other originating from the pars distalis, which enters the systemic circulation only. Whether this portal LH could modulate the output of pars distalis gonadotropes in sheep is unknown, but it should be noted that an ultrashort loop mechanism for LH appears to exist in the rabbit (46). Alternatively, the high levels of LH in portal blood may reflect the activity of a retrograde blood flow system from the pars distalis to the hypophyseal portal system (42).

It should be noted, however, that the high LH concentrations in portal blood may not be physiological and could result from the lesion made in the zona tuberalis area through which the portal vessels course. A lesion in the zona tuberalis area may cause inappropriate release of LH from surrounding gonadotropes during the collection of portal blood. It is also noteworthy that although our study argues strongly against the existence of a short loop feedback system for LH in the ewe, it was not possible to demonstrate unequivocally that icv LH accessed potential neural targets, and some caution in interpreting our data is, therefore, needed. Nevertheless, icv LH should reach any putative neural sites of action for three reasons. First, exogenous LH is still detectable in CSF up to 5 h after icv injection, and thus, this gonadotropin is not rapidly removed or bound to proteins that would prevent it from accessing putative neural targets. Second, CSF is in relatively free communication with the extracellular fluids of the brain parenchyma (21). Third, studies using rats and mice suggest that an icv administrated peptide as large as horseradish peroxidase is able to penetrate the neural tissue surrounding the cerebral ventricles (20). In addition, our LH surge studies suggest strongly that this gonadotropin does not have a GnRH terminal site of action, because the median eminence, which lacks a blood-brain barrier (21), would be exposed to the high portal LH levels that decline before corresponding GnRH concentrations fall. It must also be noted that our study does not preclude the possibility that a short loop feedback system for LH could operate in different steroidal environments. In this respect, if a short loop feedback system existed and was already operative, then icv LH may not have had an added effect in the ovariectomized ewe due to the inherently high LH levels of this model. However, as no evidence of a short loop feedback system could be detected during the estradiol-induced surge, this caveat seems improbable.

In conclusion, the present study suggests that a short loop feedback system for LH does not exist in the ovariectomized ewe, because when this gonadotropin was administered into the third ventricle, it did not modulate the pituitary release of LH into the peripheral circulation. Furthermore, the extremely low levels of LH in CSF suggest that despite the very high LH concentrations present in portal blood, little LH is able to enter the adjacent third ventricle or, probably, the hypothalamus.


   Acknowledgments
 
We thank Dr. Yves Combarnous and the NIH for the generous gifts of purified LH, Sandra Dye for performing some of the RIAs, and Dr. Jane Robinson for constructive comments on this paper.


   Footnotes
 
1 This work was supported by a Biological and Biotechnological Sciences Research Council/Institut National de la Recherche Agronomique grant (to D.C.S.). Back

2 St. Catharine’s College Research Fellow. Back

Received March 24, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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