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Endocrinology Vol. 142, No. 12 5212-5219
Copyright © 2001 by The Endocrine Society

NEUROENDOCRINOLOGY

Role of Endogenous Opioid Peptides in Mediating Progesterone-Induced Disruption of the Activation and Transmission Stages of the GnRH Surge Induction Process

T. A. Richter1, D. S. Spackman, J. E. Robinson, S. Dye, T. G. Harris, D. C. Skinner2 and N. P. Evans

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

Address all correspondence and requests for reprints to: Dr. N. P. Evans, Department of Veterinary Preclinical Studies, University of Glasgow Veterinary School, Bearsden Road, Glasgow, United Kingdom G61 1QH.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
How progesterone blocks the E2-induced GnRH surge in females is not known. In this study we assessed whether the endogenous opioid peptides (EOPs) that mediate progesterone negative feedback on pulsatile GnRH secretion also mediate the blockade of the GnRH surge. We treated ovariectomized ewes with physiological levels of E2 and progesterone to stimulate and block the GnRH surge, respectively, using LH secretion as an index of GnRH release. A pilot study confirmed that blocking opioidergic neurotransmission with the opioid receptor antagonist, naloxone (NAL; 1 mg/kg·h, iv), could prevent the suppression of pulsatile LH secretion by progesterone in our model. By contrast, antagonizing EOP receptors with NAL did not restore LH surges in ewes in which the E2-induced GnRH surge was blocked by progesterone treatment during the E2-dependent activation stage (Exp 1) of the GnRH surge induction process. However, in ewes treated with progesterone during the E2-independent transmission stage (Exp 2), NAL partially restored blocked LH surges, as indicated by increased fluctuations in LH that, in some cases, resembled LH surges. We conclude, therefore, that the EOPs that mediate progesterone negative feedback on pulsatile GnRH secretion are not involved in blockade of activation of the E2-induced GnRH surge by progesterone, but do appear to be part of the mechanism by which progesterone disrupts the transmission stage.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE ABILITY OF E2 to stimulate the preovulatory gonadotropin surge in the ewe and other mammals is regulated by progesterone. In the natural estrous cycle, the preovulatory gonadotropin surge is stimulated during the follicular phase by an elevation in E2 concentrations that occurs when progesterone concentrations are low (1). By contrast, elevations in peripheral E2 concentrations that accompany follicular growth during the luteal phase, although of comparable magnitude to those seen during the follicular phase, are unable to stimulate a gonadotropin surge due to the presence of elevated progesterone concentrations (2, 3, 4). The ability of progesterone to block the preovulatory gonadotropin surge is the basis of many oral contraceptives available to women today (5), but the mechanism by which progesterone blocks the preovulatory gonadotropin surge is not fully understood.

In rodents, progesterone prevents ovulation by acting centrally as well as at the level of the pituitary gonadotropes to block both the GnRH and LH surges (6, 7). However, in sheep and primates, the blockade of the LH surge is primarily due to progesterone acting centrally to inhibit the GnRH surge (8, 9, 10). Because GnRH neurons do not appear to express steroid receptors (11, 12, 13, 14), progesterone is thought to act on GnRH neurons indirectly via a system(s) of progesterone-receptive interneurons.

In a physiological model of the GnRH-LH surge induction process in ewes, developed in our and other laboratories (15, 16, 17, 18), it is possible to identify three distinct stages: 1) activation, which requires exposure to elevated E2 concentrations for 5–10 h; 2) an E2-independent stage during which the stimulatory E2 signal is transmitted to the GnRH neurosecretory system; and 3) the release of a surge of GnRH that stimulates a LH surge, which is also E2 independent and occurs 15–20 h after the start of the E2 signal (15, 19, 20) (Fig. 1Go). We have demonstrated that luteal phase levels of progesterone can block the E2-induced GnRH surge by 1) preventing activation of the E2-responsive positive feedback system (21), and 2) disrupting transmission, thereby preventing the stimulation of GnRH neurons by the activated positive feedback system (20) (Fig. 1Go). The latter action is limited to the early portion of the transmission phase (20), and other groups have shown that the later portion of the transmission phase is sensitive to disruption by stress (22).



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  		Figure 1. Theoretical model for the GnRH-LH surge induction process in the ewe. The three stages, namely activation (I), transmission (II), and surge (III), are illustrated relative to the time course of a normal E2-induced (E alone) surge of GnRH (gray line) and LH (black line, closed circles), and GnRH (shaded area) and LH (black line, open circles) levels after progesterone administration (E+P) during the activation and transmission stages. The gray bar indicates the period during progesterone administration has been shown to block expression of the surge. The open bars indicate the periods during which the opioid antagonist NAL was administered in the present study. All GnRH and LH data were obtained from the same ewe over consecutive artificial estrous cycles generated as in Materials and Methods. Based on Refs. 15 19 20 , and 34 .

 
Based upon the effects of endogenous opioid peptides (EOPs) on tonic GnRH and LH secretion in several species (23, 24, 25, 26, 27, 28, 29, 30), EOPs are one of the most likely candidate interneuronal systems that mediate the blockade of the GnRH surge by progesterone. Initial studies by Currie and co-workers (31, 32) suggested that the EOPs were not involved in the blockade of the GnRH surge by progesterone, because prolonged infusion of ewes with the EOP receptor antagonist, naloxone (NAL), failed to restore ovulation during a progesterone treatment regimen that prevented ovulation (31, 32). However, in light of the insights that our ovine model of the surge induction process has recently given into the inhibitory effects of progesterone on the GnRH surge, it is possible that the studies by Currie et al. (31, 32) might have failed to detect a differential involvement of EOPs in disruption of the activation and transmission stages of the surge induction process. In addition, chronic exposure to NAL and consequent up-regulation of opioid tone might have obscured more subtle progesterone-dependent opioid effects. In the present study we took advantage of the temporal delineation of the "windows" of steroid receptivity available in our model to assess whether EOPs that mediate progesterone negative feedback on pulsatile LH secretion are also involved in the specific inhibitory effects of progesterone upon the activation (Exp 1) and transmission (Exp 2) stages of the GnRH surge induction process.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
All experiments were conducted on adult Poll Dorset ewes in the breeding season (August–February). Ewes were maintained at the Babraham Institute (Cambridge, UK) under standard husbandry conditions, and all animal procedures were conducted in accordance with Home Office regulations (PPL 80/1037). The jugular veins were catheterized for the collection of blood samples and infusion of drugs. During experimentation, ewes were restrained individually in pens in which they could move forwards and backwards freely, but could not turn around.

Experimental model
Ewes were ovariectomized at least 2 wk before experimentation, and midluteal phase (background) levels of E2 were maintained [~1 pg/ml, 1-cm sc SILASTIC brand capsule (Dow Corning Corp., Midland, MI) containing crystalline E2]. Luteal phase levels of progesterone (2–4 ng/ml) were generated with two intravaginal progesterone-releasing devices (33) (CIDRs, InterAg, Hamilton, New Zealand). Artificial luteal phases were 10 d long. Approximately 24 h after CIDR withdrawal to simulate luteolysis, an artificial follicular phase was generated by administering an incremented concentration of E2 (5–8 pg/ml, four 3-cm sc SILASTIC brand capsules) (16, 19). In our laboratory, a relatively short (5–10 h) exposure to these increased E2 concentrations stimulates GnRH and LH surges after 15–20 h (20, 34, 35).

As in other studies in our laboratory (20, 34, 35), we used LH secretion as an index of GnRH secretion. This is based on the fact that LH release is directly dependent upon GnRH (36, 37, 38, 39), with the result that GnRH and LH secretion are parallel during most physiological situations, including pulsatile secretion (40, 41). Moreover, the amplitude and time of onset of the GnRH and LH surge is the same (1, 15, 37, 40, 42) (Fig. 1Go), although GnRH concentrations remain elevated for a longer duration than LH concentrations (37, 39) (Fig. 1Go). Finally, progesterone has been shown in ewes to block the LH surge by acting centrally to inhibit the GnRH surge, rather than at the level of the pituitary, since exogenous GnRH can stimulate LH release in animals treated with progesterone to block the LH surge (3).

Pilot experiment
The aim of the pilot study was to determine whether NAL could prevent the progesterone-induced inhibition of pulsatile LH release. The study design is presented in Fig. 2Go. Two groups of ewes with luteal phase levels of E2 (~1 pg/ml) were treated with a luteal phase concentration of progesterone (2–4 ng/ml) for 10 h. Group 1 (n = 6) received no further treatment, but group 2 (n = 6) received an infusion of NAL (1 mg/kg·h) from h 5–10 of the 10-h progesterone treatment period. Blood samples were collected at 15-min intervals for 5 h before progesterone treatment and during the period of NAL treatment to assess pulsatile LH secretion.



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  		Figure 2. Effect of NAL infusion on inhibition of pulsatile LH secretion by progesterone in the pilot study. A, LH secretion profiles in a representative ewe from each of the two treatment groups (n = 6/group). Filled circles represent LH pulses. Ewes in group 1 and 2 had background levels of E2 (E; solid line) and were treated with progesterone (5–15 h; gray bar). Only ewes in group 2 were also treated with NAL (10–15 h; black bar). B, Mean (±SE) LH pulse frequency and mean (±SE) LH concentrations in both groups 5 h before P treatment (0–5 h; open bars) and during the last 5 h (10–15 h; solid bars) of P treatment (group 1) or P plus NAL treatment (group 2). **, P < 0.01; ***, P < 0.001 (by t test).

 
Exp 1: can NAL prevent the blockade of activation by progesterone?
The aim of this experiment was to determine whether NAL could prevent the ability of progesterone to specifically block activation of the E2-induced surge. Three groups of animals with luteal phase levels of E2 (~1 pg/ml) were exposed to incremented (follicular phase) E2 concentrations (5–8 pg/ml) for 10 h to initiate the LH surge induction process. Positive controls (group 1; n = 8) received E2 alone and were expected to exhibit a LH surge. Negative controls (group 2; n = 8) received luteal phase levels of progesterone (2–4 ng/ml) at the same time as the E2 increment and were expected not to surge. Group 3 (n = 7) was treated simultaneously with incremented E2, progesterone, and NAL (1 mg/kg·h). Jugular blood samples were collected hourly to monitor LH surges, and additional, frequent (every 15 min) samples were collected during the midsurge period (18–24 h after the E2 increment) to assess the effects of NAL on tonic LH secretion. The study design is presented in Fig. 3Go.



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  		Figure 3. Experimental design and results for Exp 1. Ovariectomized ewes were treated with exogenous E2 (E) and progesterone (P) after a 10-d artificial luteal phase (see Materials and Methods). Upper panels, Cartoons of the experimental design and associated treatments for group 1–3. A, Positive controls (n = 8) were treated with incremented E for 10 h (0–10 h; solid line). B, Negative controls (n = 8) were treated with 10 h E and 10 h of P (0–10 h; gray bar). C, A third group (n = 7) was treated as for group 2, but also received an infusion of NAL concurrent with the P treatment (0–10 h; 1 mg/kg·h; solid bar). Lower panels, Mean (±SE) LH secretion profiles for each group. In group 1 (A), the open symbols are data for the single positive control ewe that failed to exhibit an LH surge in response to the E2 increment. The insets are LH profiles spanning the midsurge period (18–24 h after E increment) for a representative ewe from each group (note the difference in scale for group 1). Arrowheads indicate LH pulses. **, P < 0.01 for incidences of LH surges in group 1 vs. groups 2 and 3, by Fisher’s exact test.

 
Exp 2: can NAL prevent the blockade of transmission by progesterone?
The aim of this experiment was to determine whether NAL could prevent the blockade of transmission by progesterone. The design of Exp 2 was similar to that of Exp 1, except that ewes were exposed to 8 h of E2 to stimulate an LH surge, as this had been shown in a previous artificial estrous cycle to be sufficient to stimulate an LH surge in all of the animals included in this study (data not shown). The same animals were used in Exp 2A and 2B.

Exp 2A: early transmission stage
Positive controls (group 1; n = 6) were treated with incremented E2 for 8 h. Negative controls (group 2; n = 7) received the same E2 treatment, followed immediately by a 5-h period of progesterone treatment (i.e. early transmission stage; see Fig. 1Go). Group 3 (n = 6) received the same treatment as the negative controls, but was infused with NAL (1 mg/kg·h) during the 5-h progesterone treatment period. Blood samples were collected hourly to monitor LH surges, and frequent (every 12 min) samples were collected for 2 h before and 2 h after the start of the NAL infusion to assess the effects of NAL on tonic LH secretion. The study design is presented in Fig. 4Go.



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  		Figure 4. Experimental design and results for Exp 2A. Upper panels, Cartoons of the experimental design and associated treatments for group 1–3. A, Positive controls (n = 6) were treated with incremented E2 (E) for 8 h (0–8 h; solid line). B, Negative controls (n = 7) were treated with 8 h of E, followed by 5 h of progesterone (P; 8–13 h; gray bar). C, A third group (n = 6) was treated as described for group 2, but also received an infusion of NAL concurrent with the P treatment (8–13 h; 1 mg/kg·h; solid bar). Lower panels, Mean (±SE) LH secretion profiles for each group. The inset in C shows the detailed LH profile of a representative NAL-treated ewe. *, P < 0.05 for incidences of LH surges in group 1 vs. groups 2 and 3; by Fisher’s exact test.

 
Exp 2B: throughout the transmission stage
Ewes were exposed to two of three treatments over two consecutive artificial estrous cycles in a randomized cross-over design, such that no animal received the same treatment twice. The three treatments were similar to those of Exp 2A; namely, 1) positive controls (group 1; n = 11 treatment cycles) were treated with incremented E2 for 8 h; 2) negative controls (group 2; n = 12 treatment cycles) received the same E2 treatment, followed immediately by a 5-h period of progesterone treatment during the early transmission stage; and 3) group 3 (n = 11 treatment cycles) received the same treatment as the negative controls, but was infused with NAL (1 mg/kg·h) for 5 h during the progesterone treatment period and for 5 h after progesterone removal, such that NAL was infused throughout the transmission stage (see Fig. 1Go). Hourly and frequent blood samples were collected and processed as in Exp 2A. The study design is presented in Fig. 5Go.



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  		Figure 5. Experimental design and results for Exp 2B. Upper panels, Cartoons of the experimental design and associated treatments for group 1–3. A, Positive controls (n = 11 treatment cycles) were treated with incremented E2 (E; 0–8 h; solid line). B, Negative controls (n = 12 treatment cycles) were treated with 8 h E, followed by 5 h of progesterone (P; 8–13 h; gray bar). C, A third group (n = 12 treatment cycles) was treated as for group 2, but also received an infusion of NAL (1 mg/kg·h) from 8–18 h (solid bar). Lower panels, Mean (±SE) LH secretion profiles. The insets in C show the detailed LH profiles of a representative NAL-treated ewe that either exhibited surge-like LH release (no. 55) or increased pulsatile LH release (no. 112). ***, P < 0.001 for incidences of LH surges in group 1 vs. groups 2 and 3, by Fisher’s exact test.

 
LH assays
LH concentrations were measured in duplicate 100-µl aliquots of plasma using a previously described, double antibody RIA (43) with LH standard NIDDK oLH-I-3 and antiserum NIDDK anti-oLH-1 (A. F. Parlow, NIDDK, Torrance, CA). Plasma samples for each experiment were assayed separately. The interassay and intraassay coefficients of variation for the combined assays were 8.4% and 16.9%, respectively. Sensitivity averaged 0.78 ng/ml.

Data analysis
LH surges were identified using the statistical criteria of Richter et al. (34). The onset of a surge was defined as the time corresponding to the first LH sample in a continuous series of at least three hourly samples that was greater than the mean ± 2 SD of the preceding LH values. The end of a surge was defined as the time corresponding to the last LH sample in a continuous series of at least three samples that were smaller than the mean ± 2 SD of the presurge (baseline) LH values.

LH pulses were detected using an algorithm based on the Munro pulse detection program (Zaristo Software, Haddington, Scotland, UK). To be defined as a pulse, an LH value had to be: 1) greater than the baseline (defined as the mean ± 0.5 SD of all values), 2) greater than the preceding value, and 3) followed by a series of at least two progressively smaller values.

Statistics
For the pilot study, pulse frequencies and mean LH concentrations between treatments within each group were compared using a paired t test. For the remaining experiments, mean LH concentrations during surges were compared using ANOVA with Tukey’s post test, and incidences of LH surges in different treatment groups were compared using Fisher’s exact test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Pilot experiment: can NAL prevent progesterone-induced inhibition of pulsatile LH release?
There was a significant decrease in LH pulse frequency (n = 6; by t test, P < 0.01) and mean LH secreted (n = 6; by t test, P < 0.01) after progesterone treatment (Fig. 2AGo), whereas there was no change in these parameters compared with the pretreatment period in ewes treated with NAL at the same time as progesterone (n = 6; t test, P > 0.05 for both parameters; Fig. 2BGo).

Exp 1: can NAL prevent the blockade of activation by progesterone?
E2 stimulated an LH surge in seven of eight positive controls (Fig. 3AGo), whereas progesterone blocked activation of the surge in all (n = 8) negative controls (Fig. 3BGo). Infusion of NAL did not prevent the blockade of activation by progesterone (no surges in seven ewes; Fig. 3CGo). LH release during the surge was not obviously pulsatile (Fig. 3AGo, inset), but LH pulses were evident in progesterone-treated ewes (Fig. 3Go, B and C, insets).

Exp 2: can NAL prevent the blockade of transmission by progesterone?
Exp 2A: early transmission stage. E2 stimulated LH surges in all (n = 6) positive control animals (Fig. 4AGo), whereas treatment with progesterone during the early transmission stage blocked the LH surge in all (n = 7) negative controls (Fig. 4BGo). Concurrent treatment with NAL and progesterone did not restore the LH surge in any (n = 6) ewes (Fig. 4CGo). LH concentrations in the NAL-treated group, however, were elevated, relative to negative control levels, due to transient fluctuations in LH concentrations that began at approximately the same time as the onset of the LH surge in positive controls (Fig. 4CGo, inset).

Exp 2B: throughout the transmission stage. LH surges were stimulated by E2 in 10 of the 11 positive control cycles (Fig. 5AGo). Treatment with progesterone during the early transmission stage blocked the LH surge in all negative controls (n = 12 treatment cycles; Fig. 5BGo). Infusion of NAL throughout the transmission stage resulted in elevated LH release in all animals (n = 12 treatment cycles) during the time of the LH surge in positive controls, which was significantly greater than LH release in the negative controls, but lower than LH levels in the positive controls (n = 35; by ANOVA, P < 0.05 for group 2 vs. 3, P < 0.01 for group 1 vs. 3). LH responses in the ewes treated with NAL throughout the transmission stage could be divided into two distinct types (Fig. 5CGo, inset). The LH responses in 7 of the 12 treatment cycles consisted of a sustained increase from baseline that occurred at approximately the same time as the onset of surges in the positive controls and increased to reach a peak (ewe 55; Fig. 5CGo). This pattern of LH secretion met the statistical criteria required for classification as a surge, but the decrease in LH concentrations observed after LH levels peaked was not sustained, and the mean amplitude of the LH increase in these animals was significantly lower than that in positive controls (n = 15; by ANOVA, P < 0.001). LH concentrations in the 5 remaining NAL-treated ewes also increased, but the increase was irregular and suggestive of increased pulsatile secretory activity, rather than sustained release as occurs during a surge (ewe 112; Fig. 5CGo).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Elevated progesterone concentrations prevent stimulation of the preovulatory GnRH surge by E2, but the mechanism(s) that underlies the blockade of the GnRH surge remains unknown. Because EOPs mediate the inhibition of pulsatile GnRH secretion by progesterone (44), EOPs have been hypothesized to also mediate blockade of the GnRH surge. In the present study we assessed whether EOPs mediate the progesterone-dependent blockade of specific stages of the GnRH surge induction process by determining whether inhibiting opioidergic neurotransmission with the EOP receptor antagonist, NAL, could restore LH surges in ewes treated with progesterone to block either activation or transmission of a surge-inducing E2 signal. Whereas antagonizing the EOPs failed to restore surges in ewes in which the surge had been blocked by progesterone treatment during the activation stage (Exp 1), ewes treated with NAL during the transmission stage (Exp 2) exhibited increased LH secretion. In some ewes treated with NAL and progesterone throughout the transmission stage (Exp 2B), the pattern of LH secretion resembled reduced magnitude LH surges. These observations suggest that the endogenous opioidergic systems that mediate progesterone negative feedback on pulsatile GnRH secretion are not involved in the blockade of surge activation by progesterone, but are partially responsible for the progesterone-induced disruption of the transmission stage of the surge induction process.

The fact that normal LH surges could not be fully restored by antagonizing EOP receptors with NAL in the present study suggests that both the inhibitory effects of progesterone on GnRH secretion are multimodal and that EOPs are differentially involved in mediating the blockade of the surge by progesterone. Specifically, progesterone clearly acts via opioidergic neural systems to inhibit pulsatile GnRH (and thus LH) secretion, as evidenced by the inability of progesterone to inhibit pulsatile LH release in NAL-treated ewes in the present study. By contrast, progesterone does not act via EOPs to prevent activation of the estrogen-responsive positive feedback system, because NAL treatment failed to restore LH surges in ewes treated during the activation stage. However, the observation that NAL treatment partially restored surges in ewes in which progesterone disrupted the transmission stage suggests that this facet of the progesterone-induced blockade of the surge appears to be partially mediated by opioids.

The surges in LH secretion observed in some NAL-treated animals in Exp 2 suggest that the inhibitory effects of progesterone during the transmission stage are mediated through several different neurotransmitter systems, at least one of which is opioidergic. Thus, antagonizing the activity of only one such system (in this case, the opioids) would not completely prevent the blockade of the surge by progesterone, but would result in surges of reduced magnitude, as observed in Exp 2B. Evidence to support the idea that neurotransmitters besides opioids mediate blockade of the GnRH surge by progesterone includes the fact that increasing the concentration of opioids alone cannot block the surge (45, 46). In addition, progesterone-receptive neurons have been shown to release several other neurotransmitters that have been implicated in the regulation of gonadotropin secretion, including glutamate, dopamine, somatostatin, neurotensin, substance P, NPY, and {gamma}-aminobutyric acid (47, 48, 49, 50, 51, 52). Unfortunately, although there is evidence that some of these neurotransmitters inhibit pulsatile GnRH and LH secretion (see Refs. 53, 54, 55 for review), whether they are involved in mediating the blockade of the surge by progesterone has not been investigated. It is also possible the blockade of E2 positive feedback by progesterone is not only mediated by progesterone-responsive interneuronal systems, but that progesterone might also act directly in the E-receptive neurons to prevent their activation by E2. In this regard, progesterone treatment during the activation stage has been shown to prevent cellular activation throughout the hypothalamus (34), but whether this inhibition occurs specifically in the E2-receptive cells that stimulate the GnRH surge is not known.

There are several alternative explanations for the inability of NAL to fully restore LH surges in progesterone-treated ewes in the present study. First, progesterone might stimulate the release of opioid peptides that act at receptors that are not sensitive to NAL. This is unlikely, however, because NAL and its derivatives are nonspecific opioid receptor antagonists (56, 57) that act at each of the three main opioid receptor types (µ, {delta}, and {kappa} receptors) (58). Furthermore, NAL can reverse the effects of nonspecific (e.g. ß-endorphin) (59) and opioid receptor-specific agonists, such as fentanyl (µ-selective) (60), U50,488 ({kappa}-selective) (61), and (D-Ala2,D-Leu)-enkephalin ({delta}-selective) (62). Second, NAL may have failed to prevent the blockade of the surge by progesterone if the dose (1 mg/kg·h) was too low to fully antagonize progesterone-induced opioidergic neurotransmission. However, this dose has been shown to be extremely effective in disinhibiting progesterone negative feedback on GnRH secretion in similar studies (63, 64) and was clearly sufficient to completely prevent progesterone-inhibited pulsatile LH secretion in the current study. Furthermore, even lower doses of NAL than that used in the present study, (e.g. 0.5 mg/kg·h) (65) are still capable of antagonizing progesterone-inhibited LH secretion.

In conclusion, this study indicates that the EOPs that mediate the inhibition of pulsatile GnRH secretion by progesterone are not the principal type of neurotransmitter invoked by progesterone to prevent either activation of the positive feedback system by E2 or transmission of a surge-inducing signal from E2-responsive cells to the GnRH system. The observations that antagonizing opioidergic neurotransmission 1) prevented progesterone negative feedback on pulsatile LH secretion, 2) did not prevent progesterone blocking the surge by disrupting activation, and 3) partially restored GnRH surges in ewes in which transmission has been disrupted by progesterone, imply a multimodal action of progesterone on GnRH neurosecretion, wherein the effects of progesterone on pulsatile secretion and the E2-induced preovulatory surge are mediated by different neural mechanisms.


   Acknowledgments
 
Our thanks to Martin White, Andrew Dady, Rachel Forsdike, James Taylor, Will Unsworth, and Arthur Davis for assistance with the animals and surgery. We are grateful to Dr. A. F. Parlow (NIDDK) for donation of the LH antiserum.


   Footnotes
 
This work was supported by the BBSRC, a Wellcome Vacation Scholarship (VS/96/MIS/006; to D.S.S.), the Cambridge Commonwealth Trust, National Research Foundation, South Africa, and the Trustees of the Elsie Ballot Memorial Scholarship (to T.A.R.). Preliminary reports appeared in J Reprod Fertil Abstract Series 18 (1996) and 25 (2000).

1 Present address: Wisconsin Regional Primate Research Center, University of Wisconsin, 1223 Capitol Court, Madison, Wisconsin 53717-1299. Back

2 Present address: Department of Clinical Veterinary Science, University of Bristol, Langford House, Langford, Somerset, United Kingdom BS40 5DU. Back

Abbreviations: EOP, Endogenous opioid peptides; NAL, naloxone.

Received May 16, 2001.

Accepted for publication August 21, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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