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1 /97/$03.00/0 Vol. 138, No. 12 Endocrinology Printed in U.S.A. Copyright 1997 by The Endocrine Society Estradiol Requirements for Induction and Maintenance of the Gonadotropin-Releasing Hormone Surge: Implications for Neuroendocrine Processing of the Estradiol Signal* N. P. EVANS, G. E. DAHL, V. PADMANABHAN, L. A. THRUN, AND F. J. KARSCH Reproductive Sciences Program (N.P.E., G.E.D.), and Departments of Physiology (F.J.K.), Biology (L.A.T.), and Pediatrics (V.P.), University of Michigan, Ann Arbor, Michigan ABSTRACT Two experiments were performed to examine the temporal requirements of the estradiol signal for the GnRH and LH surges in the ewe. Hypophyseal portal and jugular blood (to measure GnRH and LH, respectively) were sampled from ewes set up in an artificial follicular phase model. After progesterone withdrawal to simulate luteolysis, circulating estradiol was raised to a preovulatory level by inserting estradiol implants, which then were removed at different times to vary estradiol signal duration. The objective of the first experiment was to assess the effect of withdrawing estradiol at surge onset on development and maintenance of the GnRH/LH surges. Removal of estradiol, before surge onset, neither altered the LH surge in relation to that induced when the estradiol stimulus was maintained nor affected stimulation of a massive and sustained GnRH surge that outlasted the LH surge by many hours. Continued estradiol treatment, however, did prolong the GnRH surge. In the second experiment, the estradiol stimulus was shortened to test the hypothesis that estradiol need not be present for the whole presurge period to induce GnRH/LH surges. Ewes received estradiol either up to the time of surge onset (21 h) or for periods equivalent to the last 14 h, the last GENERATION of the preovulatory LH surge is of paramount importance to fertility in females of all mammalian species. In many spontaneous ovulators, the increase in circulating estradiol concentrations during the follicular phase of the estrous or menstrual cycle initiates a cascade of neuroendocrine events that culminates in generation of the Received June 9, Address all correspondence and requests for reprints to: F. J. Karsch, Reproductive Sciences Program, University of Michigan, 300 North Ingalls Building, Room 1101 SW, Ann Arbor, Michigan * Preliminary reports appear in the abstracts of the Annual Meeting of the Society for the Study of Reproduction 1992 and the Journal of Reproduction and Fertility Abstract Series No. 15, Supported by USDA , NIH-HD-18337, and HD-18258, and the Office of the Vice President for Research at the University of Michigan. Support was also provided by the Sheep Research, Standards and Reagents, Data Analysis, and Administrative Core Facilities of the Center for the Study of Reproduction (NIH P30-HD-18258). Present address: Department of Veterinary Physiology, University of Glasgow Veterinary School, Bearsden Rd., Glasgow G61 1QH, Scotland, United Kingdom. Present address: Department of Animal and Avian Sciences, University of Maryland, College Park, Maryland Present address: Department of Internal Medicine, Division of Endocrinology and Metabolism, 5570 MSRB II, University of Michigan, Ann Arbor Michigan h, or the earliest7hofthe21-h signal. Shortening the signal to 14 h did not reduce its ability to stimulate a full GnRH surge, but it did reduce the amplitude of the resultant LH surge. Further shortening of the signal to 7 h, however, produced a mixed response. Most animals (8 of 10 combining the two 7-h groups) did not express GnRH surges. In the two ewes that did, GnRH surge amplitude and duration were again within the range observed with the 21-h estradiol signal, but the LH response was greatly reduced. These results indicate that, once the GnRH/LH surges of the ewe have begun, elevated estradiol is not required for surge maintenance. Development of a full GnRH surge requires elevated estradiol for only a portion of the presurge period. More prolonged exposure to estradiol, however, is needed to maximize pituitary responsiveness to GnRH. Since the estradiol signal for the GnRH surge is relatively short (7 14 h) and temporally located well in advance of the surge itself, these results are consistent with the hypothesis that estradiol is required only to activate the steroid-responsive neuronal elements and not for progression of the signal from these elements to the actual surge process of GnRH release. (Endocrinology 138: , 1997) preovulatory LH surge (1 3). In producing this surge, estradiol acts at both the pituitary gland to enhance responsiveness to GnRH (4 8) and hypothalamus to stimulate a large and sustained GnRH surge (9 13). At the pituitary level, estradiol acts directly upon gonadotropes to induce synthesis of GnRH receptors (14 17). At the hypothalamic level, however, the actions of estradiol are believed to be indirect. GnRH neurons, themselves, do not appear to contain classic nuclear estradiol receptors (18, 19) but reside in areas that are dense in other estradiol receptive neural phenotypes (20). Generation of the GnRH surge, therefore, most likely results from activation of estradiol-receptive neurons that are either linked to GnRH neurons directly or via one or more interneurons. Despite the importance of increased estradiol in stimulating the GnRH surge, estradiol concentrations do not remain elevated throughout the period of the natural preovulatory GnRH surge. In the ewe, for example, circulating estradiol concentrations decrease abruptly within 4 h after onset of the GnRH and LH surge (2, 21, 22). Yet, the GnRH surge itself can persist for an additional h in this species (12, 22). This temporal separation between the preovulatory GnRH surge and the estradiol signal suggests that the GnRH neu- 5408

2 ESTROGEN SIGNAL FOR GnRH SURGE 5409 rosecretory system need only be exposed to estradiol during the period that the estrogen-sensitive neuronal elements are activated, i.e. a period in advance of the GnRH surge. Once the GnRH surge begins, however, estradiol would no longer appear necessary to sustain the surge pattern of release. In the experimental model we and others have used extensively to study regulation of the GnRH surge (artificial follicular phase model), elevated estradiol is typically maintained throughout the surge (12, 23 25). This model has been criticized as being nonphysiological, possibly producing artifactual results, because the estradiol signal is not withdrawn at the time of surge onset, as occurs naturally (26). The first goal in this study, therefore, was to refine the model and assess the effect of withdrawing the estradiol signal at surge onset on development and maintenance of the GnRH surge. Having established that the massive and sustained GnRH surge was still produced when estradiol was withdrawn at surge onset, our second goal was to assess the duration of the estradiol signal needed to induce and sustain a full GnRH surge. General Materials and Methods All experiments were conducted during the midbreeding season (Exp. 1, November-December, 1992; Exp 2, November, 1994) on adult Suffolk ewes maintained under normal husbandry conditions at the Sheep Research Facility in Ann Arbor (latitude N). Our studies were performed in sheep, as this is one of the few species in which GnRH can be monitored directly in pituitary portal blood of animals that are fully conscious and not physiologically compromised. Procedures for ovariectomy, delivery of estradiol and progesterone by means of Silastic implants (Dow Corning, Midland, MI), and for remote automated sampling of hypophyseal portal blood are described elsewhere (27 30). After portal blood collection, ewes were euthanized with a barbiturate overdose (Beuthanasia, Schering-Plough Animal Health Corps, Kenilworth NJ), and the pituitary glands were inspected to determine the site and extent that the portal vasculature was cut to establish sufficient flow for sample collection. All procedures were approved by the Committee for the Use and Care of Animals at the University of Michigan. Exp 1 In this experiment, we tested the effect of withdrawing the estradiol signal at surge onset on development and maintenance of the GnRH surge. As variation in surge timing relative to estradiol implant addition is less within an animal over repeated artificial cycles than between animals in any one particular cycle, all animals were run through two artificial follicular phases, the first of which determined the time of LH surge onset for each ewe. This information was then used in the second artificial follicular phase to determine the time of estradiol implant removal. The design was as follows. Fourteen midluteal phase ewes were ovariectomized and immediately treated with a 1-cm sc Silastic capsule containing estradiol (27) and two 4 7 cm sc Silastic packets containing progesterone (28). These implants maintain serum concentrations of estradiol and progesterone equivalent to those observed in the midluteal phase of the estrous cycle, approximately 1 pg/ml and 3 ng/ml, respectively (31). Approximately 1 week later, when luteolysis would have occurred, progesterone implants were removed simulating luteal regression. Sixteen hours later, four 3-cm estradiol implants were inserted sc to simulate the preovulatory rise in serum estradiol concentrations (estradiol implants presoaked in water for 24 h to prevent an initial burst of steroid release (27)). This treatment regimen raises estradiol levels to approximately 8 pg/ml (31) and induces preovulatory-like surges of GnRH and LH approximately 20 h after estradiol addition (12) (note, the model used in this study was modified in that two rather than three progesterone packets were used for progesterone delivery; this treatment regimen produces circulating progesterone concentrations within the range seen during the natural luteal phase). During this first artificial follicular phase, the times of LH surge onset were determined for each ewe from hourly samples of jugular blood collected over a 20-h period beginning 10 h after placement of the four estradiol implants. Within 24 h after completion of blood sampling, these estradiol implants were removed and, after a further 24 h, progesterone implants were reinserted to generate a second artificial luteal phase. During this artificial luteal phase, animals underwent surgery for placement of the portal blood collection apparatus (30). Approximately 16 days after their insertion, progesterone implants were removed, and the animals were allocated to two groups for a second artificial follicular phase: controls (n 6), estradiol implants retained throughout the expected duration of the GnRH surge; experimental (-E, n 8), peak follicular phase estradiol implants removed approximately 2 h before the expected start of the GnRH surge (onset estimated from the first artificial follicular phase). Hourly samples of pituitary portal and jugular blood were collected for 35 h starting 5 h before the expected GnRH/LH surge. Pituitary portal blood was collected into 5 ml m bacitracin (Sigma, St. Louis, MO) in ice-cold saline to inhibit endopeptidase activity. Exp 2 In this experiment, we tested the hypothesis that, to induce the GnRH surge, estradiol need be present only during a certain limited time during the presurge period. The approach was similar to that of Exp 1. Twenty ewes were allocated to four groups as shown in Fig 1. Controls received the estradiol stimulus until the approximate time of GnRH surge onset (21 h, n 5). Experimental ewes were split into three groups and received an estradiol stimulus either during a period equivalent to the last 14 h of estradiol treatment in controls (14 h, n 5), the last 7 h (7L, n 5), or the earliest7hofestradiol treatment (7E, n 5). Pituitary portal and jugular blood were sampled hourly for 36 h starting 5 h before the expected start of the GnRH surge, estimated from LH responses in a previous artificial follicular phase (data not shown). Peripheral blood was collected at selected time points before, during, and after estradiol treatment to characterize the experimentally produced rise in circulating estradiol. FIG. 1. Design of Exp 2 depicted relative to the time of the addition of the peak follicular phase estradiol implants in the control group. P, Expected profile of serum progesterone concentrations; OVX, time of ovariectomy. Hatched bars indicate the time of treatment with peak follicular phase estradiol. The solid bar indicates the time during which hourly samples of portal and peripheral blood were collected.

3 5410 ESTROGEN SIGNAL FOR GnRH SURGE Endo 1997 Vol 138 No 12 Assays GnRH concentrations were measured in a previously described RIA (12, 29). Samples (750 l containing 680 l portal plasma and 70 l bacitracin) were extracted in methanol, and duplicate aliquots of the extract were assayed. To minimize the effect of between-assay variation, all samples for a given ewe were measured in a single assay. Intraassay variation (six assays), as determined by the median variance ratio of assay replicates (32), averaged 0.045; assay sensitivity averaged 0.11 pg/tube. LH was measured in duplicate aliquots of plasma ( l) using a modification (33) of a previously described RIA (34, 35) and is expressed in terms of NIH-LH-S12. Mean inter- and intraassay coefficients of variation (CV) were 7.25% and 8.01%, and assay sensitivity averaged 0.14 ng/tube. Estradiol concentrations were estimated in duplicate diethyl ether extracts of 200 l of plasma using a modification (36) of the Serono Diagnostics Estradiol MAIA assay (Serono-Baker Diagnostics Inc., Allentown, PA). Intraassay CV (six assays) at 50% bound was 12%, and assay sensitivity averaged 0.36 pg/ml. Data analysis GnRH values are expressed as rate of collection (picograms per min) rather than concentration. This minimizes potential errors caused by contamination of portal blood with either peripheral blood or cerebrospinal fluid or errors due to changes in sample volume that may occur after lifting and lowering of the ewe s head. In both experiments, presurge baselines for GnRH and LH were calculated as the mean of the first four samples, unless a consistent trend was seen for hormone concentrations to increase, in which case the mean of the first three samples was used. Surge onset was defined as the time GnRH/LH rose above two times the presurge baseline and remained at or above this level for a minimum of 4 h. The end of the surge was defined as the time GnRH/LH fell to values that remained below twice the presurge baseline. Surge duration was the interval between the start and end of the surge. If the surge had not ended (as defined above) when sampling was terminated, surge duration was taken as the interval between surge onset and the end of the collection. Surge magnitude (GnRH and LH) was taken as the highest point assayed. GnRH and LH surge magnitude and duration were compared by Student s t tests (control vs. -E in Exp 1, and controls (21 h) vs. 14 h in Exp 2). Peak estradiol concentrations in Exp 2 were compared across groups by ANOVA. After ANOVA, mean comparisons were made between control and treatment groups using Dunnett s t test. Results Exp 1 All animals exhibited a clear LH surge in the first artificial follicular phase in response to elevated estradiol (data not shown). Information on timing of this surge in each ewe was subsequently used to determine the timing of estradiol implant removal in the second artificial follicular phase. Mean GnRH and LH responses in the second artificial follicular phase are depicted in Fig. 2. In the -E group, estradiol implants were removed at the expected onset of the GnRH surge (actually removed at or shortly before surge onset in six of eight animals and just after surge onset in the remaining two ewes). On average, implants were removed 45 min before surge onset (range 5to 2 h, individual times shown as filled circles in bottom panel Fig. 2). Estradiol induced GnRH and LH surges in all animals of both groups. There were no group differences in either LH surge amplitude or duration (Fig. 3a). Mean GnRH surge amplitude did not differ significantly between treatments (Fig. 3b). In both control and -E groups, GnRH remained elevated well beyond the end of the LH surge. GnRH surge duration, however, was significantly (P 0.05) longer in the control relative to the -E group (Fig. 3b). Although the GnRH surge had ended in seven of the eight -E animals by approximately 20 h after surge onset, values were still elevated in FIG. 2. Mean GnRH and LH responses after estradiol treatment in Exp 1, aligned and plotted relative to the beginning of GnRH surge, for the control (top) and the -E (bottom) groups. Mean ( SEM) GnRH concentrations are shown by the open circles. The shaded area depicts the mean ( SEM) LH concentrations. The hatched bar in each graph denotes the time of estradiol treatment. In panel B, the hatched bar reflects the mean time of estradiol treatment; individual times of estradiol implant removal are shown by the large filled circles. FIG. 3. Mean ( SEM) LH (a) and GnRH (b) surge characteristics observed in Exp 1. Solid bars represent data obtained from the control group; open bars represent the -E group. *, P 0.05 all control ewes upon termination of the experiment ( 30 h after surge onset, Fig. 2). Exp 2 Mean GnRH and LH responses in the control (21 h) and 14 h groups are shown in Fig. 4, along with the mean estradiol

4 ESTROGEN SIGNAL FOR GnRH SURGE 5411 FIG. 4. Mean GnRH, LH, and estradiol concentrations observed in the control and 14 h groups in Exp 2. In each panel, the GnRH and LH data are aligned and plotted relative to the start of the GnRH surge. Mean ( SEM) GnRH concentrations are shown by the open circles. The shaded area depicts the mean ( SEM) LH concentrations. The time of estradiol treatment is indicated in each graph by the hatched bar (positioned relative to the mean time of estradiol treatment). Mean ( SEM) estradiol concentrations are shown by solid squares and dashed lines (plotted relative to the time of estradiol treatment). FIG. 5. Mean ( SEM) LH (a) and GnRH (b) surge characteristics observed in Exp 2. Open bars represent data obtained from the control group treated with estradiol for 21 h; closed bars depict the 14 h group. Data obtained from the two ewes that surged after treatment with estradiol for 7 h (one each for 7E and 7L) are shown as the dotted bars. **, P concentrations (dashed line). In both groups, estradiol concentrations increased within 1 2 h of addition of the four 3-cm implants, to levels that did not differ significantly between groups, and remained elevated while the implants were in place (indicated by the hatched bar). Within 1 h after removal of the four estradiol implants, circulating estradiol concentrations started to fall precipitously back to the basal level ( 1 pg/ml) provided by the remaining 1-cm estradiol implant. Estradiol exposure for either 14 or 21 h induced GnRH and LH surges in all animals. In every ewe, the GnRH surge began coincident with the LH surge and outlasted the LH surge by many hours. No differences were noted between these two groups in either GnRH surge amplitude or duration (Fig. 5b). Further, there was no difference in the duration of the LH surge between the two treatments (Fig. 5a). LH surge amplitude, however, was significantly (P 0.01) reduced in the 14 h animals relative to the controls (Fig. 5a). Time to surge onset was consistent in both groups relative to the start of the estradiol signal ( 21 h) but differed with regard to the interval from estradiol implant removal. Surges began around the time of implant removal in the control group but approximately 7 h after implant removal in the 14 h ewes (Fig. 4). Data for the two groups of ewes treated with estradiol for 7 h are plotted in Fig. 6. As in the control and 14 h groups, peripheral estradiol increased and decreased rapidly upon implant addition and removal. Peak estradiol concentrations in the two 7 h groups did not differ from each other (7E: 8.5 FIG. 6. Plasma GnRH, LH, and estradiol concentrations in the 7E and 7L groups of Exp 2. Data are plotted relative to the time of estradiol implantation. Mean ( SEM) GnRH and LH concentrations for the four animals in each group that did not surge are shown by the shaded area. Data for the one animal that did respond in each group are shown by the open symbols. Time of estradiol treatment is indicated by the hatched bar; mean ( SEM) estradiol concentrations are depicted by solid squares and dashed lines. 2.8 pg/ml, 7L: pg/ml), or the 14 h group ( pg/ml). The peak estradiol concentration achieved in the 7L group, however, was statistically lower (P 0.05) than in the 21-h controls ( pg/ml).

5 5412 ESTROGEN SIGNAL FOR GnRH SURGE Endo 1997 Vol 138 No 12 Reduction in the duration of estradiol exposure in the 7E and 7L groups resulted in an absence of surge generation in most animals. Only one of five ewes in each group responded to the estradiol stimulus with a GnRH surge. (Fig. 6 depicts group means for nonresponders coplotted with values for the single responder for both the 7E and 7L groups.) The single animal that responded in the 7E group (Ewe 3) mounted a GnRH surge quantitatively similar to that seen in the control group, but the pattern was unusual in that the GnRH surge was temporarily interrupted approximately 4 h after its onset. Of interest, the LH response in this ewe was barely evident (Figs. 5a and 6). The ewe that responded in the 7L group (Ewe 17) had a GnRH surge within the range of that seen in the controls and an LH surge that, although small relative to the controls, was similar to the LH responses in the 14 h group (Figs. 5a and 6). The ability of this one animal to surge was not due to a relatively high level of estradiol, as concentrations in this ewe were actually less than in the others of this group that did not mount a GnRH/LH surge. The GnRH and LH responses in the two ewes that expressed a surge in response to a 7-h estradiol treatment began approximately 21 h after the addition of estradiol, much as in the control and 14 h groups. Thus, the interval from withdrawal of the estradiol stimulus to surge onset was approximately 14 h (Fig. 6). Discussion The preovulatory LH surge in the ewe is induced by the antecedent rise in estradiol, which acts both at the pituitary gland to enhance responsiveness to GnRH (4, 7, 8, 37) and centrally to stimulate the release of a surge of GnRH that can persist for h (10 12). During the natural cycle, circulating estradiol concentrations begin to decrease approximately 4 h after the start of the surge and are basal within 12 h (2, 21, 22), well before the end of the GnRH surge. This temporal relationship suggests that estradiol is required to initiate the GnRH surge but not to sustain the surge once it begins. The results of the first experiment provides experimental support for this inference. A large and sustained GnRH surge was produced in the artificial follicular phase model whether estradiol was withdrawn around the time of surge onset or maintained throughout the duration of the surge. Of interest, not all laboratories have consistently observed a large and sustained estradiol-induced GnRH surge in the ewe (26). It has been suggested (26) that the reported differences between laboratories can be attributed to an artifact of the extended period of estradiol treatment in the artificial follicular phase model used extensively by our group. The results of Exp 1 argue strongly against this proposal. The amplitude of the GnRH and LH surges observed in this experiment, whether the estradiol signal was withdrawn or maintained, resembled those of the preovulatory surges we observed in intact animals (22). Although removal of the estrogenic stimulus at the time of surge onset was without effect on both GnRH surge amplitude and the existence of an extended GnRH surge, it is noteworthy that maintenance of the estradiol signal (control group of Exp 1) did prolong the duration of the GnRH surge compared with that seen in the -E group. This extension, however, was not sustained indefinitely, as GnRH values were declining in all animals in which estradiol was maintained but had not reached baseline when sampling ended. This eventual decline in GnRH in the face of continuously elevated estradiol could reflect desensitization/refractoriness of the hypothalamus to the stimulatory estradiol signal or depletion of the hypothalamic stores of releasable GnRH. In the second experiment, the period of exposure to estradiol was further shortened to either 14 or 7 h, and the LH responses were compared with those in animals receiving estradiol for 21 h (i.e. when the estradiol stimulus was terminated at or near GnRH surge onset). Abbreviation of the estradiol signal to 14 h neither diminished its ability to stimulate a GnRH surge nor altered the GnRH surge characteristics. Further shortening of the signal to 7 h, however, produced a mixed response. Most animals (eight of 10) did not respond to this greatly shortened estradiol signal with a GnRH surge. In the two animals that did respond, however, the observed GnRH surges had an amplitude and duration within the range of surges produced by exposure to estradiol for longer periods (14 and 21 h). In all animals that responded to the 7-h and 14-h estradiol treatments, the GnRH surge developed and persisted many hours after removal of the stimulatory signal. For example, with the 14-h signal, the GnRH surge began approximately 7 h and ended approximately 25 h after the fall in circulating estradiol. Thus, to stimulate and indeed maintain the extended GnRH surge, elevated estradiol need not be present for the entire presurge period. Collectively, the results of the two experiments lend insight into the mechanism for estradiol activation of the GnRH neurosecretory system. Our finding that estradiol is not needed for the whole presurge period is consistent with the view that elevated estradiol is required only during the time the estradiol-receptive neuronal system is activated. Neuroanatomical studies have demonstrated that the vast majority of GnRH neurons of sheep, like other mammals, do not contain detectable amounts of nuclear estradiol receptors (18, 19). It has thus been proposed that the influence of estradiol on GnRH neuronal activity is mediated by a system of interneurons. As such, the GnRH neurosecretory system is composed of GnRH neurons that are linked to estradiolreceptive neurons either directly or by means of at least one set of interneurons. Given this organization, the stimulatory effect of estradiol would be expected to occur in advance of the surge when the estradiol-receptive neurons are activated. According to this logic, estradiol would not necessarily be required during the period of the increased GnRH secretion that makes up the surge. Our finding that estradiol is not needed for the entire presurge period is consistent with the view that the steroid is needed only to activate the estradiolresponsive neuronal elements and not for progression of the signal from these elements to the surge process of GnRH release. In addition to its effects at the hypothalamus, estradiol also has important actions at the level of the pituitary in LH surge induction (4 6, 8, 37). Our study reinforces the importance of the pituitary actions of estradiol and suggests that the temporal requirements for the pituitary and hypothalamic

6 ESTROGEN SIGNAL FOR GnRH SURGE 5413 actions of estradiol differ. Although the shortened estradiol signals (14 and 7 h) induced GnRH surges that were not different from those seen in the control group in this study [or of natural surges or estradiol-induced surges in the artificial follicular phase model of previous studies (12, 22)], the LH surges produced in response to the 7- and 14-h estradiol stimuli were greatly reduced in amplitude. Presumably, this was due to decreased pituitary responsiveness to GnRH. The sensitizing effect of estradiol on the pituitary is well documented in a variety of species including the rat (5), sheep (4, 7, 8, 37), cow (38), and monkey (6). This action reflects the ability of estradiol to enhance GnRH receptor number and GnRH receptor messenger RNA (mrna) in pituitary cells (14 17). We are not aware of previous information concerning the duration or pattern of estradiol exposure required to induce these changes. The differing hypothalamic and pituitary requirements for estradiol to generate full GnRH and LH surges may be interpreted in two ways. First, the duration and timing of the estrogenic signal required by the hypothalamus and the pituitary may differ, the pituitary requiring a longer exposure than the hypothalamus. Second, the sensitizing effects of estradiol at the level of the pituitary may be short lived, in which case the steroid would have to be elevated immediately before onset of the LH surge for pituitary responsiveness to be maximized. The former possibility is supported by the finding of Clarke and Cummins (7), who observed that peak pituitary responsiveness to GnRH did not develop until h after treatment with a bolus of estradiol. In considering the different pituitary responses to the 14- and 21-h estradiol signals, we must question whether the amplitude of the induced estradiol rise was a factor. In the 21-h control group, the circulating estradiol concentration increased abruptly for the first 1 2 h after implantation and then gradually increased over the remainder of the 21-h treatment period (Fig. 4). Since the estradiol stimulus was terminated 7 h earlier in the 14-h group, the perceived amplitude of the estradiol signal may have been less than in the 21-h controls, thus reducing pituitary responsiveness to GnRH. Two points argue against this possibility. First, there was no significant difference in peak estradiol concentration between groups. Second, within the range of plasma estradiol concentrations in the two groups, no difference in amplitude of the LH surge was observed in a previous extensive dose-response study (31). Thus, the group difference in LH surge amplitude was most likely due to the different durations rather than size of the estradiol signal. Interestingly, in both experiments, the timing of the GnRH surge was consistent relative to the time of addition of the estradiol implants and bore no relation to the time of withdrawal of the stimulus. The GnRH and LH surges invariably began approximately 21 h after estradiol was applied, irrespective of both the duration of estradiol treatment and the time of the stimulus relative to progesterone withdrawal. This observation has two implications. First, although we do not know how long the activational response to estradiol persists or how long estradiol remains bound to its receptor, our findings are consistent with the concept that estradiol triggers a neuronal cascade. This cascade requires a finite time for the signal to be perceived and transmitted to the GnRH release apparatus and for the surge to occur. Second, the temporal separation of surge onset from the drop in estradiol (Exp 2) and the fact that full amplitude surges can develop when estradiol is not withdrawn (Exp 1), indicate that the preovulatory GnRH/LH surge results from the elevation in estradiol rather than from the drop in estradiol that coincides with surge onset. The surge, therefore, is not a rebound response from the intense negative feedback of the previously high level of estradiol. Finally, it is interesting to note that neither the reduction in estradiol-signal length from 21 h to 14 h, nor the further reduction to 7 h in those animals that mounted a surge, led to a change in amplitude or duration of the GnRH surge. In the artificial follicular phase model, therefore, the GnRH surge appears to be an all-or-none phenomenon, which can then be extended further if stimulation is continued as in the controls of Exp 1. This contrasts dramatically with generation of the LH surge by the pituitary which, in the ewe, requires continued stimulation by GnRH (39). Removal of the stimulatory GnRH signal at any point along the LH surge abruptly terminates the LH surge. This differing temporal dependency of the LH surge upon GnRH, and the GnRH surge upon estradiol, again may reflect the direct vs.indirect means by which the two secretory systems are activated. In summary, once the GnRH surge of the ewe has begun, continued exposure to elevated estradiol is not required for surge maintenance, although such treatment may prolong the GnRH surge. The period during which the estradiol signal is required to stimulate a GnRH surge is relatively short, between 7 h and 14 h, and temporally located well in advance of the GnRH surge itself. The ability of a temporally remote estrogenic signal to induce a subsequent GnRH surge complements the hypothesis that the surge-inducing effects of estradiol are mediated indirectly via one or more sets of interneurons that are linked to GnRH neurons, and that estradiol need only be present when the estradiol-responsive neurons are activated. Although 7 14 h of exposure to elevated estradiol is sufficient for the GnRH surge, a longer estradiol stimulus is essential for maximal pituitary response and development of a full amplitude LH surge. Acknowledgments We are grateful to Mr. Douglas D. Doop and Mr. Gary McCalla for help with the animal experimentation; Ms. Barbara H. Glover and Ms. J. Van Cleeff for assistance with the performance of the experiment and the RIAs; and Gordon D. Niswender and Leo E. Reichert, Jr., for supplying assay reagents. References 1. Freeman M 1993 The neuroendocrine control of the ovarian cycle of the rat. In: Knobil E, Neill JD (eds) The Physiology of Reproduction, ed 2. Raven Press, New York, vol 2: Goodman RL 1993 Neuroendocrine control of the ovine estrous cycle. In: Knobil E, Neill JD (eds) The Physiology of Reproduction, ed. 2. Raven Press, New York, vol 2: Hotchkiss J, Knobil E 1993 The menstrual cycle and its neuroendocrine control. In: Knobil E, Neill JD (eds) The Physiology of Reproduction, ed 2. Raven Press, New York, vol 2: Reeves JJ, Arimura A, Schally AV 1971 Pituitary responsiveness to purified luteinizing hormone-releasing hormone (LH-RH) at various stages of the estrous cycle in sheep. J Anim Sci 32: Aiyer MS, Chiappa SA, Fink G 1974 A priming effect of luteinizing hormone releasing factor on the anterior pituitary gland in the female rat. J Endocrinol 62:

7 5414 ESTROGEN SIGNAL FOR GnRH SURGE Endo 1997 Vol 138 No Knobil E 1974 On the control of gonadotropin secretion in the rhesus monkey. Recent Prog Horm Res 30: Clarke IJ, Cummins JT 1984 Direct pituitary effects of estrogen and progesterone on gonadotropin secretion in the ovariectomized ewe. Neuroendocrinology 39: Kaynard AH, Malpaux B, Robinson JE, Wayne NL, Karsch FJ 1988 Importance of pituitary and neural actions of estradiol in induction of the LH surge in the ewe. Neuroendocrinology 48: Sarkar DK, Fink G 1979 Effects of gonadal steroids on output of luteinizing hormone releasing factor into pituitary stalk blood in the female rat. J Endocrinol 80: Clarke IJ 1988 Gonadotrophin-releasing hormone secretion (GnRH) in anoestrous ewes and the induction of GnRH surges by oestrogen. J Endocrinol 117: Caraty A, Locatelli A, Martin GB 1989 Biphasic response in the secretion of gonadotrophin-releasing hormone in ovariectomized ewes injected with oestradiol. J Endocrinol 123: Moenter SM, Caraty A, Karsch FJ 1990 The estradiol-induced surge of gonadotropin-releasing hormone in the ewe. Endocrinology 127: Xia L, Van Vugt D, Alston EJ, Luckhaus J, Ferin M 1992 A surge of gonadotropin-releasing hormone accompanies the estradiol-induced gonadotropin surge in the rhesus monkey. Endocrinology 131: Clarke IJ, Cummins JT, Crowder ME, Nett TM 1988 Pituitary receptors for GnRH in relation to changes in pituitary and plasma gonadotropins in ovariectomized hypothalamo/pituitary-disconnected ewes. II. A marked rise in receptor number during acute feedback effects of estradiol. Biol Reprod 39: Gregg DW, Nett TM 1989 Direct effects of estradiol-17 on the number of gonadotropin-releasing hormone receptors in the ovine pituitary. Biol Reprod 40: Brooks J, Currie RJW, Struthers WJ, McNeilly AS 1994 Involvement of oestradiol, inhibin, and GnRH in the regulation of GnRH receptor mrna expression during the follicular phase of the sheep oestrous cycle. J Reprod Fertil Abstr Ser 13: Hamernik DL, Clay CM, Turzillo A, Van Kirk EA, Moss GE 1995 Estradiol increases amounts of messenger ribonucleic acid for gonadotropin-releasing hormone receptors in sheep. Biol Reprod 53: Herbison AE, Robinson JE, Skinner DC 1993 Distribution of estrogen receptor-immunoreactive cells in the preoptic area of the ewe: co-localization with glutamic acid decarboxylase but not luteinizing hormone-releasing hormone. Neuroendocrinology 57: Lehman MN, Karsch FJ 1993 Do gonadotropin-releasing hormone, tyrosine hydroxylase-, and -endorphin-immunoreactive neurons contain estrogen receptors? A double-label immunocytochemical study in the Suffolk ewe. Endocrinology 133: Herbison AE 1995 Neurochemical identity of neurones expressing oestrogen and androgen receptors in the sheep hypothalamus. J Reprod Fertil Suppl 49: Karsch FJ, Foster DL, Legan SJ, Ryan KD, Peter GK 1979 Control of the preovulatory endocrine events in the ewe: interrelationship of estradiol, progesterone, and luteinizing hormone. Endocrinology 105: Moenter SM, Caraty A, Locatelli A, Karsch FJ 1991 Pattern of gonadotropinreleasing hormone (GnRH) secretion leading up to ovulation in the ewe: existence of a preovulatory GnRH surge. Endocrinology 129: Kasa-Vubu JZ, Dahl GE, Evans NP, Moenter SM, Padmanabhan V, Thrun LA, Karsch FJ 1992 Progesterone blocks the estradiol-induced gonadotropin discharge in the ewe by inhibiting the surge of gonadotropin-releasing hormone. Endocrinology 131: Caraty A, Antoine C, Delaleu B, Locatelli A, Bouchard P, Gautron J-P, Evans NP, Karsch FJ, Padmanabhan V 1995 Nature and bioactivity of gonadotropinreleasing hormone (GnRH) secreted during the GnRH surge. Endocrinology 136: Evans NP, Dahl GE, Mauger DT, Padmanabhan V, Thrun LA, Karsch FJ 1995 Does estradiol induce the preovulatory GnRH surge in the ewe by inducing a progressive change in the mode of operation of the GnRH neurosecretory system? Endocrinology 136: Clarke IJ 1993 Variable patterns of gonadotrophin releasing hormone secretion during the estrogen-induced luteinizing hormone surge in ovariectomized ewes. Endocrinology 133: Karsch FJ, Dierschke DJ, Weick RF, Yamaji T, Hotchkiss J, Knobil E 1973 Positive and negative feedback control by estrogen of luteinizing hormone secretion in the rhesus monkey. Endocrinology 92: Karsch FJ, Legan SJ, Hauger RL, Foster DL 1977 Negative feedback action of progesterone on tonic luteinizing hormone secretion in the ewe: dependence on the ovaries. Endocrinology 101: Caraty A, Locatelli A 1988 Effect of time after castration on secretion of LHRH and LH in the ram. J Reprod Fertil 82: Caraty A, Locatelli A, Moenter SM, Karsch FJ 1994 Sampling of hypophyseal portal blood of conscious sheep for direct monitoring of hypothalamic neurosecretory substances. In: Levine J, Conn PM (eds) Pulsatility in Neuroendocrine Systems: Methods in Neuroscience. Academic Press, San Diego, CA, vol 20: Goodman RL, Legan SJ, Ryan KD, Foster DL, Karsch FJ 1981 Importance of variations in behavioural and feedback actions of oestradiol to the control of seasonal breeding in the ewe. J Endocrinol 89: Duddleson WG, Midgley AR, Niswender GD 1972 Computer program sequence for analysis and summary of radioimmunoassay data. Comput Biomed Res 5: Hauger RL, Karsch FJ, Foster DL 1977 A new concept for control of the estrous cycle of the ewe based on the temporal relationships between luteinizing hormone, estradiol and progesterone in peripheral serum and evidence that progesterone inhibits tonic LH secretion. Endocrinology 101: Niswender GD, Midgley AR, Reichert LE 1968 Radioimmunologic studies with murine, ovine and porcine luteinizing hormone. In: Rosenberg E (ed) Gonadotropins. GERON-X, Los Altos, CA, pp Niswender GD, Reichert LE, Midgley AR, Nalbandov AV 1969 Radioimmunoassay for bovine and ovine luteinizing hormone. Endocrinology 84: Evans NP, Dahl GE, Glover BH, Karsch FJ 1994 Central regulation of pulsatile GnRH secretion by estradiol during the period leading up to the preovulatory GnRH surge in the ewe. Endocrinology 134: Hooley RD, Baxter RW, Chamley WA, Cumming IA, Jonas HA, Findlay JK 1974 FSH and LH response to gonadotropin-releasing hormone during the ovine estrous cycle and following progesterone administration. Endocrinology 95: Padmanabhan V, Kesner JS, Convey EM 1978 Effects of estradiol on basal and luteinizing hormone releasing hormone (LHRH)-induced release of luteinizing hormone (LH) from bovine pituitary cells in culture. Biol Reprod 18: Evans NP, Dahl GE, Caraty A, Padmanabhan V, Thrun LA, Karsch FJ 1996 How much of the GnRH surge is required for generation of the LH surge in the ewe? Duration of the endogenous GnRH signal. Endocrinology 137: