Endocrine Alterations That Underlie Endotoxin-Induced Disruption of the Follicular Phase in Ewes 1

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1 BIOLOGY OF REPRODUCTION 62, 45 5 (2000) Endocrine Alterations That Underlie Endotoxin-Induced Disruption of the Follicular Phase in Ewes 1 Deborah F. Battaglia,,5 Holly B. Krasa, 5 Vasantha Padmanabhan, 4,5 Catherine Viguié, 5 and Fred J. Karsch 2,,5 Departments of Physiology and Pediatrics, 4 Reproductive Sciences Program, 5 University of Michigan, Ann Arbor, Michigan ABSTRACT Two experiments were conducted to investigate endocrine mechanisms by which the immune/inflammatory stimulus endotoxin disrupts the follicular phase of the estrous cycle of the ewe. In both studies, endotoxin was infused i.v. (00 ng/kg per hour) for 26 h beginning 12 h after withdrawal of progesterone to initiate the follicular phase. Experiment 1 sought to pinpoint which endocrine step or steps in the preovulatory sequence are compromised by endotoxin. In sham-infused controls, estradiol rose progressively from the time of progesterone withdrawal until the LH/FSH surges and estrous behavior, which began 48 h after progesterone withdrawal. Endotoxin interrupted the preovulatory estradiol rise and delayed or blocked the LH/FSH surges and estrus. Experiment 2 tested the hypothesis that endotoxin suppresses the high-frequency LH pulses necessary to stimulate the preovulatory estradiol rise. All 6 controls exhibited highfrequency LH pulses typically associated with the preovulatory estradiol rise. As in the first experiment, endotoxin interrupted the estradiol rise and delayed or blocked the LH/FSH surges and estrus. LH pulse patterns, however, differed among the six endotoxin-treated ewes. Three showed markedly disrupted LH pulses compared to those of controls. The three remaining experimental ewes expressed LH pulses similar to those of controls; yet the estradiol rise and preovulatory LH surge were still disrupted. Our results demonstrate that endotoxin invariably interrupts the preovulatory estradiol rise and delays or blocks the subsequent LH and FSH surges in the ewe. Mechanistically, endotoxin can interfere with the preovulatory sequence of endocrine events via suppression of LH pulsatility, although other processes such as ovarian responsiveness to gonadotropin stimulation appear to be disrupted as well. INTRODUCTION Successful estrous or menstrual cyclicity requires a sequence of causally related events, each leading to the subsequent step and all required for the completion of one cycle and progression to the next. Immune/inflammatory challenge can profoundly disrupt this intricate hormonal in- 1 This work was supported by NIH MH-1165 and HD-187; the Sheep Research, Standards and Reagents, Data Analysis, and Administrative Core Facilities of the P0 Center for the Study of Reproduction (NIH HD ); and the Office of the Vice President for Research at the University of Michigan. Preliminary reports have appeared in the Society for Neuroscience Abstracts, Vol. 22, Part, p. 1791, 1996 and Vol. 2, Part, p. 1245, Correspondence: Fred J. Karsch, Reproductive Sciences Program, University of Michigan, 00 North Ingalls Building, Room 1101 SW, Ann Arbor, MI FAX: ; fjkarsch@umich.edu Received: 14 May First decision: 29 June Accepted: 2 August by the Society for the Study of Reproduction, Inc. ISSN: terplay and thus interfere with ovarian cyclicity. Individually, various regulatory steps have been shown to be disrupted. For example, the bacterial toxin endotoxin given repeatedly during the natural follicular phase prevents the LH surge and induces cystic follicles in cows [1]. In monkeys, repeated challenge with endotoxin during the early to mid-follicular phase interrupts the preovulatory estradiol rise, delays the LH surge, and, in some animals, suppresses subsequent luteal phase progesterone production [2]. Chronic treatment with the cytokine interleukin 1 (IL-1), a key mediator of endotoxin effects [,4], disrupts the estrous cycle of rats and suppresses GnRH gene expression as well as LH and FSH secretion [5]. Additionally, both endotoxin and IL-1 can inhibit activation of GnRH neurons in rats on the afternoon of proestrus [6,7] and thus expression of the preovulatory LH surge. Despite such investigations assessing the impact of immune challenge on individual components of the sequence of events driving cyclicity, no study has temporally linked multiple endocrine perturbations within the same individual, thus allowing identification of the pathophysiologic basis for cycle disruption. More specifically, the primary step or steps in the preovulatory cascade of endocrine events compromised by immune challenge have not been pinpointed. Of interest in this regard, endotoxin and cytokines potently suppress pulsatile GnRH and LH secretion in gonadectomized animals [8 1]. However, no study to date has put this response into a physiologic perspective by determining the effects of immune challenge on the emergence of high-frequency pulses of LH during the follicular phase. This is of particular interest since such high-frequency LH pulses are necessary to generate the preovulatory estradiol rise that, in turn, leads to subsequent steps in the preovulatory sequence [14 16]. In the present study, we conducted two experiments to investigate the pathophysiologic basis for follicular phase disruption, using endotoxin as an immune/inflammatory challenge and the ewe as our animal model. In the first experiment, we sought to pinpoint which endocrine step or steps in the preovulatory sequence are compromised by endotoxin. In the second experiment, we tested the hypothesis that endotoxin suppresses development of the high-frequency LH pulses essential to stimulate the preovulatory estradiol rise. MATERIALS AND METHODS Animal Model Experiments were conducted in two successive breeding seasons (experiment 1, Nov Dec, 1995; experiment 2, Oct Nov, 1996) on adult, ovary-intact Suffolk ewes maintained outdoors under standard husbandry conditions at the Sheep Research Facility near Ann Arbor, MI. Estrous behavior was monitored using raddled, vasectomized rams penned

2 46 BATTAGLIA ET AL. together with the ewes throughout the study. All procedures were approved by the Committee for the Use and Care of Animals at the University of Michigan. Initially, a population of animals from the general flock was given an injection of Lutalyse (PGF 2, 10 mg/ewe; Upjohn, Kalamazoo, MI) to regress corpora lutea. Those ewes exhibiting estrous behavior (marker of the LH surge) within 1 days were determined to be in approximately the same stage of the cycle. To synchronize the subsequent follicular phase, two intravaginal progesterone-releasing devices (Controlled Internal Drug Releasing [CIDR] devices; InterAg, Hamilton, New Zealand) were placed 6 7 days after estrus to maintain circulating luteal phase concentrations of progesterone following regression of the corpus luteum [17]. Withdrawal of the CIDR devices 10 days after placement (i.e., 2 days after the corpus luteum had regressed) caused progesterone to plummet and synchronized the 2- to -day follicular phase among ewes [17]. In experiment 2, ewes were given a second injection of Lutalyse several days before removal of CIDR devices to ensure that all corpora lutea had regressed. To infuse endotoxin i.v., we used programmable, battery-operated pumps (Model 6 MP; Autosyringe, Hooksett, NH) contained within backpacks, as previously described [18]. The pumps allowed continuous delivery of endotoxin with minimal restraint or disturbance to the sheep. Backpacks were placed on all sheep at least 2 days before progesterone withdrawal to allow adjustment to the devices. An intravenous jugular catheter was inserted at the time of backpack placement for infusion of endotoxin; controls received sham infusions (venipuncture but no cannula inserted, adhesive tape wrapped around neck at venipuncture site, infusion backpack strapped to sheep). Endotoxin (Escherichia coli 05:B55; Sigma, St. Louis, MO) was reconstituted in sterile saline and infused at a rate of 00 ng/kg per hour (0.16 ml/h). The endotoxin dose was chosen on the basis of observations in orchidectomized rams, in which it induced transient sickness behaviors, including fever, cough, labored respiration, diarrhea, and lethargy (personal communication, Dr. Cynthia Daley, California State University, Chico). Jugular blood was sampled by venipuncture. Body temperature was monitored by rectal thermometer. Natural photoperiodic conditions were maintained until the time of sampling, when lights were turned on briefly for nighttime blood collections. During the frequent blood sampling to evaluate LH pulsatile secretion in experiment 2, ewes were moved indoors and then released outside for the rest of the study. Experiment 1: Which Endocrine Steps in the Preovulatory Sequence Are Disrupted by Endotoxin? We first sought to pinpoint which step or steps in the preovulatory sequence of endocrine events are compromised by endotoxin. Beginning 12 h after progesterone withdrawal (time of progesterone withdrawal defined as Hour 0), ewes were either infused with endotoxin (n 6) for 26 h (i.e., Hours 12 8) or sham-infused as a control (n 8). Jugular blood was sampled at 1- to 4-h intervals for measurement of LH, FSH, estradiol, progesterone, and cortisol beginning 12 h before progesterone withdrawal and continuing until Hour 104 (4-h intervals from Hours 12 to 8 and 64 to 104; 2-h intervals from Hours 8 to 10 and 40 to 64; 1-h intervals from Hours 11 to 40). Estrus was monitored at the time of each blood sample. Rectal temperature was measured at several time points during and just after endotoxin infusion. Experiment 2: Does Endotoxin Disrupt LH Pulsatile Secretion During the Follicular Phase? We next tested the hypothesis that the disruptive effects of endotoxin could be due to a suppression of the development of high-frequency LH pulses essential for generating the preovulatory estradiol rise. The overall design was the same as in experiment 1 (n 6 endotoxin-infused ewes, n 6 sham-infused controls). Jugular blood was sampled at 4-h intervals for measurement of LH, FSH, estradiol, progesterone, and cortisol beginning 12 h before progesterone withdrawal and continuing until Hour 104 in endotoxin-treated ewes and Hour 72 in controls (end of sampling in controls was 24 h after the last control exhibited estrus). To monitor LH pulsatile secretion, jugular blood samples were taken at 5-min intervals during three -h windows of time from Hours 12 15, 24 27, and 5 8 relative to progesterone withdrawal (LH surge expected Hour 48). (Note, with respect to endotoxin infusion, that the pulse windows occurred at the beginning [Hours 0 ], middle [Hours 12 15], and end [Hours 2 26] of the 26-h endotoxin infusion.) Rectal temperature was measured before progesterone withdrawal (baseline) and at the end of each pulse window. Estrous behavior was recorded at the time of each blood sample (except during the time of high-frequency sampling). Assays LH was measured in duplicate 5- to 200- l aliquots of plasma using a modification [19] of a previously described RIA [20,21] and is expressed in terms of NIH-LH-S12. Mean intra- and interassay coefficients of variation (20 assays) were 7.% and 9.2%, respectively, and assay sensitivity for 200 l averaged 0.60 ng/ml. FSH was measured in duplicate 100- l aliquots of plasma [17] using reagents procured from the National Hormone and Pituitary Program, and is expressed in terms of NIDDK-ovine (o)fsh- 1 (AFP 5679C). Mean intra- and interassay coefficients of variation (4 assays) were 5.5% and 10.6%, respectively, and assay sensitivity averaged 0.52 ng/ml. Estradiol concentrations were determined in duplicate diethyl ether extracts of 200 l of plasma using a modification [22] of the Serono Diagnostics Estradiol MAIA assay (Serono-Baker Diagnostics Inc., Allentown, PA). Progesterone was measured in duplicate 100- l aliquots of unextracted plasma using the Coat-A-Count Progesterone kit (DPC, Los Angeles, CA), validated previously [2]. Cortisol was determined in duplicate 50- l aliquots of unextracted plasma by use of the Coat-A-Count Cortisol assay kit (DPC), previously validated for use in the sheep [8]. Plasma steroid concentrations (estradiol, progesterone, cortisol) for individual animals were determined in a single assay to minimize betweenassay variability. Within-assay variability, determined by the median variance ratios of assay replicates [24], averaged 10%, 2.8%, and 4.5%, and sensitivity averaged 0.8 pg/ml, 0.04 ng/ml, and 0.90 ng/ml for the estradiol, progesterone, and cortisol assays, respectively. Data Analysis All hormonal concentrations were log-transformed before analysis to normalize variability across a range of hormone concentrations. Effects of treatment within the same

3 ENDOTOXIN DISRUPTS THE FOLLICULAR PHASE 47 increases and decreases was set at 2.6. Level of significance was established at P FIG. 1. Mean ( SEM) serum cortisol concentrations in all control (open circles, n 8) and endotoxin-treated (closed circles, n 6) ewes in experiment 1. Follicular phase onset was synchronized by withdrawing intravaginal progesterone devices. Endotoxin (00 ng/kg/h, shading) was infused for 26 h beginning 12 h after progesterone withdrawal. Endotoxin significantly stimulated cortisol secretion (P 0.001). animal (e.g., before vs. during endotoxin) were analyzed by paired t-tests. Differences in single values between control and experimental groups (e.g., time to LH surge peak) were determined by unpaired t-tests. Two-way ANOVA with repeated measures was used to compare hormonal profiles over time between groups (interaction, group time). In experiment 2, pulses of LH were identified using the cluster pulse detection algorithm of Veldhuis and Johnson [25]. Sizes for both peak and nadir clusters were set at two samples. The value of the t statistic used to identify significant RESULTS Experiment 1: Which Endocrine Steps in the Preovulatory Sequence Are Disrupted by Endotoxin? Mean plasma progesterone just before its withdrawal ( ng/ml) was at a late luteal phase level [19,26] and not different between control and experimental groups. Cortisol remained basal in controls (overall mean, ng/ml), whereas cortisol was stimulated by endotoxin (P 0.001), with values peaking at ng/ml 4 h after starting endotoxin infusion (Fig. 1). Of interest, cortisol declined over time despite continued endotoxin treatment and approached pretreatment levels by the end of the 26-h infusion. Figure 2 illustrates the hormonal profiles and onset of estrous behavior in a representative control and 2 endotoxin-treated ewes. Controls exhibited the expected estradiol rise after progesterone withdrawal (defined as Time 0), and concurrent LH and FSH surges, which peaked at 48 h (e.g., Fig. 2a, #165). Onset of estrus was observed before the peak of the LH surge (6 h before for #165). In all endotoxin-treated ewes, the progression of follicular phase endocrine events was disrupted, although the specific nature of the disruption varied among individuals. For example, in experimental ewe #117 (Fig. 2b), estradiol continued to rise during the first 8 h after progesterone withdrawal, approaching preovulatory peak values. Within 12 h of beginning endotoxin infusion, however, the estradiol rise was interrupted, and values fell to near assay sensitivity. LH/ FSH surges and estrous behavior did not occur at the expected time in this ewe (mean SEM time to LH peak in controls depicted by solid bar along top abscissa in Fig. 2). Soon after endotoxin infusion was terminated, estradiol again rose and culminated in LH and FSH surges and estrous behavior, although these preovulatory events were all FIG. 2. Plasma LH, FSH, and estradiol profiles in a representative control (a) and two endotoxin-treated ewes (b and c) in experiment 1. Onset of estrus is indicated by the arrow. Mean ( SEM) time to LH surge peak in controls is depicted by the black bar at the top. Follicular phase onset was synchronized by withdrawing intravaginal progesterone devices. Endotoxin (00 ng/kg/h, shading) or sham infusion occurred for 26 h beginning 12 h after progesterone withdrawal.

4 48 BATTAGLIA ET AL. FIG.. Summary data for time of the LH surge peak (closed circles) and estrus onset (open circles) in all control and endotoxin-treated ewes in experiment 1. LH surge and estrous responses in individual animals are linked by the line between the open and closed circles. Follicular phase onset was synchronized by withdrawing intravaginal progesterone (P) devices. Endotoxin significantly delayed the LH surge peak and estrous expression (P 0.01). Note, one endotoxin ewe did not express estrus or the LH surge during the experiment (shaded circle). delayed relative to means in the controls (Hours 64, 68, and 60 for LH peak, FSH peak, and estrus, respectively). In experimental ewe #407 (Fig. 2c), endotoxin again disrupted the preovulatory estradiol rise and markedly delayed the LH/FSH surges and estrus (peak of LH/FSH surges at Hour 96; estrus at Hour 84). Before these surges, both LH and FSH became abnormally elevated and approached concentrations observed in ovariectomized ewes (mean values between 48 and 72 h: LH ng/ml, FSH ng/ml). Despite the elevated gonadotropin levels, estradiol remained markedly suppressed for 24 h after endotoxin infusion ended. Thereafter, circulating estradiol increased, leading to the LH surge. Of note, on several occasions FSH approached values similar to those seen during the preovulatory FSH surge. Such a marked elevation in gonadotropins following endotoxin was observed in 2 additional ewes (not illustrated). The responses in individual ewes were borne out in the overall hormonal and behavioral responses in both control and experimental groups. Two-way ANOVA with repeated measures revealed a significant difference between groups in the time course of estradiol after progesterone withdrawal (P 0.00; interaction, group time). In controls, mean plasma estradiol rose progressively from progesterone withdrawal until the time of the LH surge. In contrast, the estradiol rise was interrupted during the 0-h endotoxin infusion, restarting at varying times (4 26 h) after treatment was terminated. Figure summarizes the time to the LH surge peak and estrus onset in all control and experimental ewes. Overall, endotoxin significantly delayed the LH surge and estrous behavior (control vs. endotoxin: LH peak, 48 vs h; estrus, 41 2 vs. 68 7h;P 0.01). Of note, one endotoxin-treated ewe failed to express the LH surge or estrus during the observation period (data excluded from analysis). FIG. 4. Mean ( SEM) plasma progesterone (a), cortisol (b), body temperature (c), and estradiol (d) profiles in all control (open circles) and endotoxin-treated (closed circles) ewes in experiment 2 (n 6 ewes/ group). Follicular phase onset was synchronized by withdrawing intravaginal progesterone (P) devices. Endotoxin (00 ng/kg/h, shading) significantly stimulated progesterone, cortisol, and temperature (P 0.001), and interrupted the estradiol rise (P 0.00). Experiment 2: Does Endotoxin Disrupt LH Pulsatile Secretion During the Follicular Phase? As in experiment 1, circulating progesterone on the day of its withdrawal was at a luteal phase level ( ng/ ml) and did not differ between groups. Although progesterone plummeted similarly in both groups after the CIDR devices were removed, the time course of progesterone in control and experimental ewes differed during and following endotoxin (Fig. 4a; P by two-way ANOVA with repeated measures; interaction, group time). Specifically, progesterone remained basal in controls whereas, in experimental ewes, progesterone was stimulated by endotoxin. (This increase in progesterone was most likely of adrenal origin, as a similar rise was induced by endotoxin in ovariectomized ewes [8].) Mean cortisol values and rectal temperature remained at or near basal levels in controls, whereas both variables were significantly stimulated by endotoxin (Fig. 4, b and c; P 0.001). Two-way ANOVA with repeated measures revealed a significant group difference in the time course of circulating estradiol (Fig. 4d; P 0.00; interaction, group time). In controls, estradiol rose progressively from the time of progesterone withdrawal until the LH surge (estradiol peaked at varying times such that the composite plot obscures true peak value). In con-

5 ENDOTOXIN DISRUPTS THE FOLLICULAR PHASE 49 FIG. 5. Plasma LH, FSH, and estradiol profiles, and LH pulse patterns in a representative control ewe (a) and an endotoxin-treated ewe whose pulses were blocked (b) in experiment 2. Mean ( SEM) time to LH surge peak in controls is depicted by the black bar at the top. Follicular phase onset was synchronized by withdrawing intravaginal progesterone devices. Endotoxin (00 ng/kg/h, shading) or sham infusion occurred for 26 h beginning 12 h after progesterone withdrawal. LH pulses were monitored by 5-min jugular blood sampling at the beginning (1), middle (2), and end () of endotoxin infusion; closed circles in LH pulse windows indicate peaks of LH pulses. trast, endotoxin interrupted the estradiol rise in all ewes as it did in experiment 1, with values falling to barely detectable levels by the end of infusion. Figures 5 and 6 depict plasma estradiol, LH, and FSH concentrations (4-h samples) and LH pulse profiles (5-min samples) in a representative control and in endotoxintreated ewes; Table 1 summarizes data for all ewes. As in experiment 1, all controls exhibited an estradiol rise culminating in estrous behavior and LH/FSH surges 2 days after withdrawal of progesterone (Fig. 5a, Table 1). During all windows of 5-min sampling, controls exhibited LH pulsatile secretion similar to that previously observed in the follicular phase [27], with a frequency of 1 pulse/h (Fig. 5a, Table 1). As in experiment 1, endotoxin disrupted the follicular phase of all 6 experimental ewes, interrupting the preovulatory estradiol rise and delaying or blocking LH/FSH surges and estrous behavior (Fig. 5b and Fig. 6, a and b). In three of the ewes, endotoxin markedly suppressed LH pulses (Table 1, #6006, 6075, 6079; Fig. 5b depicts hormonal profiles in ewe #6079). LH pulses in these ewes were often absent, and those pulses that were detected usually were of small amplitude compared to controls (e.g., Fig. 5b, #6079, second pulse in window 1). None of these ewes expressed a preovulatory estradiol rise, LH/FSH surges, or estrous behavior during the observation period. In the other three ewes, endotoxin did not markedly suppress LH pulses, although a subtle suppression was evident in two of these ewes during the latter stage of the first window of frequent samples (Fig. 6, a and b). Pulse frequency was similar to FIG. 6. Plasma LH, FSH, and estradiol profiles and LH pulse patterns in two endotoxin-treated ewes whose pulses were not blocked in experiment 2. Ewe #6081 (a) exhibited delayed LH/FSH surges whereas ewe #6029 (b) did not express LH/FSH surges. Mean ( SEM) time to LH surge peak in controls is depicted by the black bar at the top. Closed circles in LH pulse windows indicate peaks of LH pulses. See legend for Figure 5 for experimental details.

6 50 BATTAGLIA ET AL. that of controls (Table 1, #6017, 6029, 6081). Two of these ewes exhibited the LH surge, although it was delayed (Table 1; example Fig. 6a, #6081). The other ewe expressing LH pulses did not exhibit the LH surge (Fig. 6b, #6029). Of interest, LH values in this ewe rose markedly after endotoxin was terminated and remained elevated (mean 12 ng/ml) at values similar to those in ovariectomized ewes [26]. FSH also became abnormally elevated in this ewe after endotoxin treatment was terminated, and no FSH surge was expressed. Despite these sustained and elevated LH and FSH levels, estradiol remained basal (Hours 48 92, Fig. 6b). Similar FSH rises were observed in other endotoxin-treated ewes, and a clear-cut FSH surge occurred in only one of them. Figure 7 illustrates the mean plasma cortisol and progesterone concentrations during endotoxin treatment in each of the 6 experimental ewes, as determined in the 4-h blood samples. Of interest, the ewes in which LH pulses were suppressed (solid circles) had greater mean plasma cortisol and progesterone levels during endotoxin infusion than the ewes that continued to express LH pulses (open circles; statistical comparisons not made because of small animal numbers). FIG. 7. Plasma cortisol (left) and progesterone (right) concentrations during endotoxin infusion (12 6 h after progesterone withdrawal) in the ewes in which LH pulses were blocked (dark circles) and the ewes that continued to express LH pulses (open circles) in experiment 2. Values in each sheep are the mean of the 4-h samples obtained during the 26-h infusion of endotoxin. TABLE 1. LH pulse frequency in -h sampling windows during the follicular phase, and the time of LH peak and estrous behavior in control and endotoxin-treated ewes. Group/ewe Hours from progesterone withdrawal LH peak Hours to Estrus Control a Endotoxin b c nd d a Values are mean SEM LH pulse frequency (pulses/h) during each window (n 6 control ewes). b Values are individual pulse frequency (pulses/h) during each window for each experimental ewe. c Indicates no expression of the LH surge or estrus during the 104 h following endotoxin. d nd, Indicates data are not reliable for this ewe because of technical problems. Effects on Subsequent Cycle In experiment 1, estrous behavior was monitored daily in the cycle following endotoxin treatment. All control and endotoxin-treated ewes expressed estrus days after the prior estrus. The interval from first to second estrus, however, was slightly but significantly shorter in ewes treated with endotoxin, suggesting an abbreviated estrous cycle (control vs. endotoxin, vs days; P 0.01). In experiment 2, plasma progesterone revealed a tendency for a shortened luteal phase in experimental ewes (number of thrice-weekly samples with progesterone elevated above 1 ng/ml; control vs. endotoxin, vs ; P 0.07). Those ewes whose follicular phase endocrine profiles were most severely disrupted by endotoxin exhibited the shortest progesterone elevations. In the extreme case, ewe #6029, only one progesterone value approached a luteal phase level ( 1 ng/ml) before the subsequent estrus. (This ewe had markedly elevated gonadotropins and no estradiol rise or LH surge during the follicular phase of endotoxin treatment; Fig. 6b.) DISCUSSION When we began this work, prior studies indicated that immune/inflammatory challenges, such as endotoxin or the cytokine IL-1, could interrupt the follicular phase of the estrous cycle in the rat, cow, and rhesus monkey [1,2,5]. Disruptive effects included impaired follicular development and a delayed or blocked preovulatory LH surge. No investigation, however, had determined the pathophysiologic basis of the hormonal alterations or identified the step or steps in the preovulatory cascade of endocrine events interrupted by immune challenge. In a recently published study in the rhesus monkey, repeated bolus injections of endotoxin during the follicular phase interfered with the preovulatory estradiol rise and prevented generation of the LH surge at the usual time [2]. This suppression of preovulatory estradiol secretion, however, was not accompanied by reduced circulating gonadotropins, at least not revealed by daily blood sampling. If anything, serum LH and FSH concentrations were increased by endotoxin. Of importance, none of the previous investigations had assessed the influence of immune challenge on the pulsatile pattern of LH secretion during the follicular phase. Although immune challenge had been shown to inhibit pulsatile GnRH and LH secretion in gonadectomized animals [8 1], comparable studies were lacking in follicular phase animals. Such a determination is important because the high-frequency pulse pattern that develops during the follicular phase produces a gonadotropin environment necessary to stimulate the preovulatory estradiol rise [14 16]. We thus embarked upon these studies in the ewe with the general goal of identifying the endocrine step(s) in the preovulatory sequence disrupted by endotoxin (experiment

7 ENDOTOXIN DISRUPTS THE FOLLICULAR PHASE 51 FIG. 8. Preovulatory endocrine events in the ewe (adapted from Goodman, [14]). Left) Hormonal secretory profiles. Right) The sequence of endocrine regulatory steps. The follicular phase begins when circulating progesterone (Prog) falls at luteolysis, initiating the following endocrine cascade: increased GnRH pulse frequency, increased LH pulse frequency, preovulatory estradiol (E) rise, GnRH surge, LH surge, ovulation. The present observations, in conjunction with prior results, provide evidence that endotoxin disrupts at least steps in the cascade, designated 2,, 4. See text for further explanations. 1) and the more specific goal of testing the hypothesis that endotoxin disrupts the follicular phase by suppressing pulsatile LH secretion (experiment 2). The findings in both experiments clearly demonstrate that endotoxin interrupted the preovulatory estradiol rise and consequently delayed or blocked the LH/FSH surge in all ewes. As soon as 4 h after onset of endotoxin treatment, circulating estradiol began to plummet. Quite to our surprise, however, endotoxin did not invariably inhibit pulsatile LH secretion. Whereas endotoxin suppressed LH pulses in half the ewes, the remaining animals continued to express LH pulses seemingly unabated. Thus, while endotoxin disrupted the follicular phase of all ewes, interfering with preovulatory estradiol secretion and delaying or blocking the LH/FSH surge, it appeared to do so by more than one mechanism. Two compelling questions immediately arise from our findings: What other mechanisms may exist by which endotoxin interrupts the follicular phase? Why does endotoxin inhibit LH pulses in some follicular phase ewes but not others? With respect to the pathophysiologic basis of follicular phase disruption, our findings allow us to consider the possibility that endotoxin may act by at least three mechanisms to alter the follicular phase (illustrated in Fig. 8). First, data in half of our animals indicate that endotoxin can act at a neuroendocrine level, potently suppressing pulsatile LH release after progesterone withdrawal (Fig. 8, step 2). The recent finding that endotoxin inhibits pulsatile GnRH secretion in ovariectomized ewes [8] suggests this suppression occurs at a hypothalamic level, although further work is needed to determine whether this holds true for follicular phase ewes. Of interest, IL-1 mrna expression is induced by endotoxin in brain regions relevant to GnRH regulation in the rat [28]. Because IL-1 can suppress GnRH release in rats [7], it is possible that this cytokine is involved in central inhibition of pulsatile LH secretion. Second, endotoxin may also exert suppressive effects at the ovarian level, impairing follicular development and/or inhibiting estradiol secretion in response to gonadotropic stimulation (Fig. 8, step ). In support of this mechanism, the estradiol rise in half our animals was disrupted despite the persistence of normal LH pulse patterns. Further, previous studies indicate that immune challenge can inhibit gonadotropin-induced steroidogenic and ovulatory responses in rats [29 1]. Third, endotoxin may compromise the LH surge system (Fig. 8, step 4). Several of our ewes initially produced an estradiol rise similar to that subsequently observed in the same animals just before the surge, and to that in controls, yet the LH surge was still delayed (Fig. 2b and Fig. 6a). Also suggesting disruption of the surge mechanism, evidence in the rat indicates that central IL-1 can inhibit the ovarian steroid-induced LH surge [2], and, in the ewe, systemic endotoxin can prevent the LH surge-generating mechanism from responding to the positive feedback estradiol signal []. Beyond the steps addressed in the present study, it is worth noting that endotoxin can cause corpus luteum regression [4] and interfere with ovulation [1] (steps 1 and 4 in Fig. 8). Taken together, these findings provide evidence that the mechanisms of follicular phase disruption are complex and may well be achieved at multiple levels. The question related to the split LH pulse response to endotoxin is more difficult to address. On the one hand, our finding that endotoxin inhibited LH pulses in some ewes but not others could reflect a methodological problem. Namely, we did not monitor LH pulse profiles continuously, and thus we might have missed a period when LH pulses were suppressed. Of interest, there was a hint of suppression late in the initial -h sampling window in 2 of the ewes in which LH pulses were judged not to be suppressed overall (Fig. 6, window 1 of both ewes). Consistent with this, the induction of IL-1 mrna in the rat brain following endotoxin appears to be time-dependent, with expression occurring relatively briefly in areas relevant to GnRH regulation [28]. In addition, the time course of endotoxin inhibition of pulsatile GnRH secretion in ovariectomized ewes is variable [8]. On the other hand, the split LH pulse response may relate to the overall pathophysiologic responses to endotoxin, which are complex and involve interactions of many homeostatic regulatory functions beyond altered neuroendocrine activity. The sum total of these responses is likely to differ among individuals depending on their genetic composition, present physiologic status, and past immune-adaptive responses. For example, we observed that the cortisol and adrenal progesterone responses tended to be greater in those ewes whose pulses were suppressed. Perhaps these adrenal responses provide a general indicator of how profoundly neuroendocrine systems are impacted by endotoxin. Further, the cortisol and/or progesterone elevations themselves may have contributed to LH pulse suppression, in which case the smaller steroid responses may not have reached a threshold for neuroendocrine suppression. Of interest, both cortisol and progesterone can inhibit progression of the follicular phase in several species including sheep [5 7]. Another question arising from the continuance of LH pulses in some ewes receiving endotoxin pertains to con-

8 52 BATTAGLIA ET AL. trasting observations in ovariectomized ewes. We have found that a bolus injection of endotoxin, at doses substantially smaller than those infused here, invariably and potently suppresses pulsatile LH secretion in ovariectomized ewes ([8] and unpublished observations). These differing observations in our studies may be related to the chronic mode of endotoxin administration used in the present studies versus the acute bolus treatment used previously. Alternatively, the presence of ovarian hormones in follicular phase ewes may protect the LH pulse-generating mechanism from immune challenge, ameliorating endotoxin suppression. In this regard, estradiol has been suggested to alter the response to central challenge with IL-1 in the rhesus monkey [8,9]. The possibility that estradiol and other ovarian hormones may modulate reproductive neuroendocrine responses to immune challenge is intriguing and worthy of further study. Several aspects of the responses we observed after termination of endotoxin infusion are of interest: 1) resumption of the preovulatory endocrine sequence, 2) hypersecretion of LH and FSH, and ) carry-over effects into the subsequent estrous cycle. Relative to resumption of the follicular phase, it is interesting to note that animals differed in the timing and degree of recovery following endotoxin. This was most evident in terms of estradiol. In some ewes, follicular phase estradiol secretion returned as soon as 4 h after endotoxin treatment was terminated, while in others, estradiol remained suppressed throughout the entire observation period (see Fig. 6, #6081 vs. 6029). This difference may reflect disruption of follicles at variable stages of development, some at stages able to recover from endotoxin and others at stages unable to do so. In the latter case, another follicle may have to mature before preovulatory estradiol production can be restored. The elevated LH and FSH levels following endotoxin in some ewes may be the consequence of the suppression of estradiol secretion and temporary release of the neuroendocrine axis from negative feedback inhibition. Of note, similar gonadotropin elevations in response to endotoxin have been observed in the rhesus monkey [2]. The most extreme case in our study was a ewe in which estradiol fell to a barely detectable level during endotoxin infusion despite continuance of seemingly normal LH pulses (Fig. 6b, #6029). After endotoxin treatment was terminated, both LH and FSH increased to values typically observed in ovariectomized ewes, but estradiol production did not recover. Perhaps this ewe did not regain ovarian responsiveness to gonadotropins, possibly because her preovulatory follicle was at a stage different from that of her partners in which estradiol secretion returned, or because no replacement follicle was available. Finally, our findings provide preliminary evidence for carry-over effects of endotoxin into the subsequent cycle. Although we did not monitor that cycle in great detail, endotoxin appears to have shortened the luteal phase and reduced progesterone production, especially in ewes whose follicular phase was most severely disrupted. This apparent influence on the subsequent luteal phase complements findings in the rhesus monkey indicating that endotoxin reduces circulating progesterone in some animals when administered during the previous follicular phase [2]. Of interest, luteal phase dysfunction in women, characterized by an inadequate quantity or duration of progesterone secretion, has been linked to alterations in gonadotropin secretion during the preceding follicular phase [40]. In conclusion, systemic challenge with the immune/inflammatory stimulus endotoxin disrupts progression of the follicular phase of the ewe, interrupting the preovulatory estradiol rise and thus blocking or delaying the LH and FSH surges. Mechanistically, our data support the hypothesis that endotoxin can act at a neuroendocrine level, suppressing the pulsatile LH secretion vital to development of the preovulatory estradiol rise and generation of the positive feedback estradiol signal. In addition, our findings suggest that endotoxin can also inhibit at the level of the ovary, preventing estradiol production in response to gonadotropic stimulation, and at the level of the LH surge-generating mechanism, interfering with response to the preovulatory estradiol rise. Prior studies have provided evidence that various types of immune challenge can impair some of these functions individually in diverse experimental models. Our findings enhance the physiological significance of this earlier work by pinpointing several key steps in the natural progression of preovulatory events that are likely to be disrupted by immune challenge, at least that provoked by endotoxin. Collectively, these alterations constitute the pathophysiologic basis of follicular phase disruption. ACKNOWLEDGMENTS The authors are indebted to Doug Doop and Gary McCalla for their expertise with the animal experimentation. We would like to thank Ms. Martha Brown for her input in the design and interpretation of the results and for technical support, Drs. Lori Thrun and Jennifer Bowen for help in conducting the experiments, and Dr. Thomas Harris for his contributions to the final formulation of the manuscript. We also thank Drs. Gordon D. Niswender and Leo E. Reichert, Jr. for supplying RIA reagents and Dr. Johannes D. Veldhuis for providing the Cluster pulse detection algorithm. 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