Two types of burst firing in gonadotrophin-releasing hormone neurones.

Gonadotrophin-releasing hormone (GnRH) neurones fire spontaneous bursts of action potentials, although little is understood about the underlying mechanisms. In the present study, we report evidence for two types of bursting/oscillation driven by different mechanisms. Properties of these different types are clarified using mathematical modelling and a recently developed active-phase/silent-phase correlation technique. The first type of GnRH neurone (1-2%) exhibits slow (∼0.05 Hz) spontaneous oscillations in membrane potential. Action potential bursts are often observed during oscillation depolarisation, although some oscillations were entirely subthreshold. Oscillations persist after blockade of fast sodium channels with tetrodotoxin (TTX) and blocking receptors for ionotropic fast synaptic transmission, indicating that they are intrinsically generated. In the second type of GnRH neurone, bursts were irregular and TTX caused a stable membrane potential. The two types of bursting cells exhibited distinct active-phase/silent-phase correlation patterns, which is suggestive of distinct mechanisms underlying the rhythms. Further studies of type 1 oscillating cells revealed that the oscillation period was not affected by current or voltage steps, although amplitude was sometimes damped. Oestradiol, an important feedback regulator of GnRH neuronal activity, acutely and markedly altered oscillations, specifically depolarising the oscillation nadir and initiating or increasing firing. Blocking calcium-activated potassium channels, which are rapidly reduced by oestradiol, had a similar effect on oscillations. Kisspeptin, a potent activator of GnRH neurones, translated the oscillation to more depolarised potentials, without altering period or amplitude. These data show that there are at least two distinct types of GnRH neurone bursting patterns with different underlying mechanisms.

Rapid nongenomic effects of oestradiol on gonadotrophin-releasing hormone neurones.

That oestradiol can have both negative- and positive-feedback actions upon the release of gonadotrophin-releasing hormone (GnRH) has been understood for decades. The vast majority of studies have investigated the effects of in vivo oestrogen administration. In the past decade, evidence has accumulated in many neuronal and non-neuronal systems indicating that, in addition to traditional genomic action via transcription factor receptors, steroids can also initiate effects rapidly via signalling cascades typically associated with the cell membrane. Here, we review work examining the rapid actions of oestradiol on GnRH neurones, addressing the questions of dose dependence, receptor subtypes, signalling cascades and intrinsic and synaptic properties that are rapidly modulated by this steroid.

Depolarising and hyperpolarising actions of GABA(A) receptor activation on gonadotrophin-releasing hormone neurones: towards an emerging consensus.

The gonadotrophin-releasing hormone (GnRH) neurones represent the final output neurones of a complex neuronal network that controls fertility. It is now appreciated that GABAergic neurones within this network provide an important regulatory influence on GnRH neurones. However, the consequences of direct GABA(A) receptor activation on adult GnRH neurones have been controversial for nearly a decade now, with both hyperpolarising and depolarising effects being reported. This review provides: (i) an overview of GABA(A) receptor function and its investigation using electrophysiological approaches and (ii) re-examines the past and present results relating to GABAergic regulation of the GnRH neurone, with a focus on mouse brain slice data. Although it remains difficult to reconcile the results of the early studies, there is a growing consensus that GABA can act through the GABA(A) receptor to exert both depolarising and hyperpolarising effects on GnRH neurones. The most recent studies examining the effects of endogenous GABA release on GnRH neurones indicate that the predominant action is that of excitation. However, we are still far from a complete understanding of the effects of GABA(A) receptor activation upon GnRH neurones. We argue that this will require not only a better understanding of chloride ion homeostasis in individual GnRH neurones, and within subcellular compartments of the GnRH neurone, but also a more integrative view of how multiple neurotransmitters, neuromodulators and intrinsic conductances act together to regulate the activity of these important cells.

Neurobiological mechanisms underlying oestradiol negative and positive feedback regulation of gonadotrophin-releasing hormone neurones.

The feedback actions of ovarian oestradiol during the female reproductive cycle are among the most unique in physiology. During most of the cycle, oestradiol exerts homeostatic, negative feedback upon the release of gonadotrophin-releasing hormone (GnRH). Upon exposure to sustained elevated oestradiol levels, however, there is a switch in the feedback effects of this hormone to positive, resulting in induction of a surge in the release of GnRH that serves as a neuroendocrine signal to initiate the ovulatory cascade. We review recent developments stemming from studies in an animal model exhibiting daily switches between positive and negative feedback that have probed the neurobiological mechanisms, including changes in neural networks and intrinsic properties of GnRH neurones, underlying this switch in oestradiol action.

Long-term recordings of networks of immortalized GnRH neurons reveal episodic patterns of electrical activity.

The CNS controls reproduction through pulsatile secretion of gonadotropin-releasing hormone (GnRH). Episodic increases in the firing rate of unidentified hypothalamic neurons have been associated with downstream markers of GnRH secretion. Whether this episodic electrical activity is intrinsic to GnRH neurons, intrinsic to other "pulse generator" neurons that drive GnRH neurons, or a combination of these is unknown. To determine if GnRH neurons display episodic firing patterns in isolation from other cell types, immortalized GnRH neurons (GT1-7 cells) were cultured on multiple microelectrode arrays. Long-term, multi-site recordings of GT1-7 cells revealed repeated episodes of increased firing rate with an interval of 24.8 +/- 1.3 (SE) min that were completely eliminated by tetrodotoxin, a sodium channel blocker. This pattern was comprised of active units that fired independently as well as coincidentally, suggesting the overall pattern of electrical activity in GT1-7 cells emerges as a network property. The A-type potassium-channel antagonist 4-aminopyridine (1 mM) increased both firing rate and GnRH secretion, demonstrating the presence of A-type currents in these cells and supporting the hypothesis that electrical activity is associated with GnRH release. Physiologically relevant episodic firing patterns are thus an intrinsic property of immortalized GnRH neurons and appear to be associated with secretion. The finding that overall activity is derived from the sum of multiple independent active units within a network may have important implications for the genesis of the GnRH secretory pattern that is delivered to the target organ. Specifically, these data suggest not every GnRH neuron participates in each secretory pulse and provide a possible mechanism for the variations in GnRH-pulse amplitude observed in vivo.

Cycles of transcription and translation do not comprise the gonadotropin-releasing hormone pulse generator in GT1 cells.

Neural control of reproduction is achieved through episodic GnRH secretion, but little is known about the molecular mechanisms underlying pulse generation. The ultradian time domain of GnRH release suggests mechanisms ranging from macromolecular synthesis to posttranslational modification could be involved. We tested if messenger RNA (mRNA) or protein synthesis are components of the pulse generator by determining the effects of transcription and translation inhibitors on episodic GnRH release from immortalized GT1-1 GnRH neurons. Time course and efficacy of transcription and translation blockade were assessed by determining the ability of specific inhibitors to block the robust, rapid induction of c-fos mRNA or protein accumulation by forskolin (10 microM). The transcription inhibitors actinomycin D (ACT-D, 20 microM) or 5,6-dichlorobenzimidazole riboside (DRB, 100 microM), or the translation inhibitors anisomycin (ANI, 10 microM) or puromycin (PUR, 10 microM) were applied to GT1-1 cells 30, 15, or 0 min before forskolin. Northern and Western blots revealed blockade of transcription and translation was rapid and essentially complete. GT1-1 cells were perifused for a 90- to 120-min control period then for 100-130 min with vehicle or inhibitor to examine pulsatile GnRH secretion. GnRH interpeak intervals, peak amplitude, and peak area were not different between control and experimental periods of cells treated with vehicle (n = 15), ACT-D (n = 10), DRB (n = 6), ANI (n = 8), and PUR (n = 6; P > 0.05). This study presents the first clear evidence that the series of reactions resulting in secretion of a GnRH pulse do not include cycles of transcription and translation. Although these mechanisms would be required to replenish components of the pulse generator, they are not integral components of this oscillator. We hypothesize that posttranslational events underlie episodic GnRH release in GT1-1 cells.

Whole-cell recordings from preoptic/hypothalamic slices reveal burst firing in gonadotropin-releasing hormone neurons identified with green fluorescent protein in transgenic mice.

Central control of reproduction is governed by a neuronal pulse generator that underlies the activity of hypothalamic neuroendocrine cells that secrete GnRH. Bursts and prolonged episodes of repetitive action potentials have been associated with hormone secretion in this and other neuroendocrine systems. To begin to investigate the cellular mechanisms responsible for the GnRH pulse generator, we used transgenic mice in which green fluorescent protein was genetically targeted to GnRH neurons. Whole-cell recordings were obtained from 21 GnRH neurons, visually identified in 200-microm preoptic/hypothalamic slices, to determine whether they exhibit high frequency bursts of action potentials and are electrically coupled at or near the somata. All GnRH neurons fired spontaneous action potentials, and in 15 of 21 GnRH neurons, the action potentials occurred in single bursts or episodes of repetitive bursts of high frequency spikes (9.77 +/- 0.87 Hz) lasting 3-120 sec. Extended periods of quiescence of up to 30 min preceded and followed these periods of repetitive firing. Examination of 92 GnRH neurons (including 32 neurons that were located near another green fluorescent protein-positive neuron) revealed evidence for coupling in only 1 pair of GnRH neurons. The evidence for minimal coupling between these neuroendocrine cells suggests that direct soma to soma transfer of information, through either cytoplasmic bridges or gap junctions, has a minor role in synchronization of GnRH neurons. The pattern of electrical activity observed in single GnRH neurons within slices is temporally consistent with observations of GnRH release and multiple unit electrophysiological correlates of LH release. Episodes of burst firing of individual GnRH neurons may represent a component of the GnRH pulse generator.

Genetic targeting of green fluorescent protein to gonadotropin-releasing hormone neurons: characterization of whole-cell electrophysiological properties and morphology.

GnRH neurons form the final common pathway for central control of reproduction, with regulation achieved by changing the pattern of GnRH pulses. To help elucidate the neurobiological mechanisms underlying pulsatile GnRH release, we generated transgenic mice in which the green fluorescent protein (GFP) reporter was genetically targeted to GnRH neurons. The expression of GFP allowed identification of 84-94% of immunofluorescently-detected GnRH neurons. Conversely, over 99.5% of GFP-expressing neurons contained immunologically detectable GnRH peptide. In hypothalamic slices, GnRH neurons could be visualized with fluorescence, allowing for identification of individual GnRH neurons for patch-clamp recording and subsequent morphological analysis. Whole-cell current-clamp recordings revealed that all GnRH neurons studied (n = 23) fire spontaneous action potentials. Both spontaneous firing (n = 9) and action potentials induced by injection of depolarizing current (n = 17) were eliminated by tetrodotoxin, indicating that voltage-dependent sodium channels are involved in generating action potentials in these cells. Direct intracellular morphological assessment of GnRH dendritic morphology revealed GnRH neurons have slightly more extensive dendrites than previously reported. GnRH-GFP transgenic mice represent a new model for the study of GnRH neuron structure and function, and their use should greatly increase our understanding of this important neuroendocrine system.

Gonadotropin-releasing hormone requirements for ovulation.

This article addresses the role of GnRH in ovulation in the context of two general models of GnRH action--deterministic and permissive. According to the deterministic model, increased GnRH secretion is required to induce the preovulatory LH surge and thus ovulation. The permissive model, in contrast, holds that GnRH secretion need not increase. Rather, the preovulatory LH surge results from enhanced sensitivity of the pituitary gland to GnRH. Studies in rodents and rabbits support the deterministic model whereas evidence in primates suggests that GnRH is permissive. Three lines of evidence are presented to support the conclusion that GnRH plays a deterministic role in sheep. First, a large GnRH surge is secreted together with the preovulatory LH surge. Second, the follicular phase increase in circulating estradiol concentration stimulates this GnRH surge by a positive feedback effect. Third, initiation of the LH surge requires an abrupt increase in GnRH, and maintenance of the LH surge requires continued GnRH support. Collectively, these observations document the fundamental importance of a GnRH surge to ovulation and generation of the estrous cycle of sheep.

Circannual alterations in the circadian rhythm of melatonin secretion.

To determine if a circadian rhythm known to be functionally related to the reproductive axis varies on a circannual basis, we monitored the circadian secretion of melatonin at monthly intervals for 2 years in four ovariectomized, estradiol-implanted ewes held in a constant short-day photoperiod. Prior to the study, ewes had been housed in a short-day (8L:16D) photoperiod for 4 years and were exhibiting circannual reproductive rhythms as assessed by serum luteinizing hormone (LH) levels. Three of the four sheep showed unambiguous deviations from the expected nocturnal melatonin secretion at two different times approximately 1 year apart. Nocturnal rises in melatonin, which usually last the duration of the dark phase, were delayed by 3-14 h or were missing. Altogether, five of the seven melatonin alterations observed in these three ewes occurred during the nadir of the circannual LH cycle. In the remaining ewe, we did not observe an altered melatonin secretory pattern during this period, and this ewe also failed to show a high amplitude circannual cycle of LH. The results provide evidence for a circannual change in the circadian rhythm of melatonin secretion. This alteration in melatonin secretion may serve as a "functional" change in daylength, and thereby may influence the expression of the circannual reproductive rhythm of sheep held in a fixed photoperiod for an extended time.

The thyroid gland is required for reproductive neuroendocrine responses to photoperiod in the ewe.

A study was conducted to determine the role of the thyroid gland in three neuroendocrine responses to photoperiod; secretion of melatonin, PRL, and LH. Ewes were thyroidectomized (THX) in midsummer or left thyroid intact, and both groups were moved indoors to artificial short days (8 h of light, 16 h of darkness) for 90 days. Thereafter, a subset of both THX and thyroid-intact ewes was challenged with long days (16 h of light, 8 h of darkness) for 120 days. The other ewes remained in short days so that neuroendocrine responses to the photoperiodic shift could be distinguished from hormonal changes that occur spontaneously. Blood was sampled twice weekly for determination of serum concentrations of LH and PRL and hourly for 48 h surrounding the photoperiodic switch for assay of melatonin. All ewes were ovariectomized and treated with constant release implants of estradiol, so that PRL and LH secretion would not be influenced by alterations in gonadal steroid secretion. There was no effect of thyroidectomy on the circadian pattern of circulating melatonin or on the change in this pattern after the shift from short to long days. Similarly, thyroidectomy did not alter the PRL response to this photoperiodic shift; long days caused PRL to increase whether the thyroid was present or absent. In marked contrast, thyroidectomy blocked the effect of long days on circulating LH, a hormone indicative of reproductive neuroendocrine activity. Specifically, long days induced a precipitous drop in LH in thyroid-intact ewes, but not in THX ewes. Thus, although the thyroid plays an obligatory role in photoperiodic inhibition of the reproductive neuroendocrine axis of ewes, it may not be required for photoneuroendocrine responses in terms of melatonin and PRL secretion. Our findings suggest that in the absence of the thyroid, the reproductive neuroendocrine axis is uncoupled from the photoperiodic influence between the pineal and the GnRH neurosecretory system.

Photoperiodic synchronization of a circannual reproductive rhythm in sheep: identification of season-specific time cues.

Seasonal reproduction in the ewe is generated by an endogenous circannual rhythm of reproductive neuroendocrine activity. Exposure to as few as 70 days of photoperiodic information a year is sufficient to synchronize the rhythm. The present study was conducted to identify which portions of the photoperiodic cycle are utilized for synchronization. For this purpose, we used pinealectomized ewes that could not respond reproductively to changes in day length. Selected photoperiodic information was provided via infusion of melatonin, a hormone that provides the neuroendocrine code for day length in this species. Melatonin was delivered according to circadian patterns. The infusion patterns were tailored to mimic those of melatonin secretion in pineal-intact ewes during one of the four seasons: winter, spring, summer, or autumn. The infusions were provided for 90 days a year during each of the three years following pinealectomy. The ewes were ovariectomized and treated with constant-release Silastic capsules containing estradiol; reproductive neuroendocrine activity was monitored by measurement of serum concentrations of LH. In the absence of exogenous melatonin, most (19 of 24) pinealectomized controls exhibited circannual LH cycles that were not in synchrony, indicating that the rhythm was free-running. Melatonin synchronized the rhythm (such that the period was 365 days and the stages of the rhythm were both concurrent among animals and in appropriate phase with the geophysical year), but not all melatonin patterns were equally effective in this regard. The most effective melatonin patterns mimicked those of secretion during summer. Spring and autumn melatonin patterns were less effective, and winter melatonin patterns were ineffective. These results support the concept that there is a seasonal specificity with regard to the photoperiodic cues that synchronize the circannual rhythm of reproductive neuroendocrine activity in the ewe. The rhythm is synchronized most effectively by long-day photoperiodic cues perceived on or around the summer solstice.

Seasonal changes in gonadotropin-releasing hormone secretion in the ewe: alteration in response to the negative feedback action of estradiol.

Two experiments were performed to test the hypothesis that there is a seasonal change in the negative feedback effect of estradiol on episodic secretion of GnRH in the ewe. The first experiment identified a specific estradiol treatment (delivered by s.c. Silastic implant) that produced a 50% decrease in the frequency of pulsatile secretion of LH in ovariectomized ewes during the anestrous season. In the second experiment, this estradiol treatment was administered to ovariectomized ewes during the mid-breeding and anestrous seasons. Separate groups of ovariectomized ewes not treated with estradiol were included during each season to test for a seasonal difference in the effect of estradiol on episodic GnRH and LH secretion. Samples of hypophyseal portal blood (for GnRH) and jugular blood (for LH) were obtained at 5-min intervals approximately one month after placement of the estradiol implants. During the breeding season, no effect of estradiol was observed on either the frequency or size of GnRH and LH pulses. During anestrus, however, estradiol produced a profound suppression of the frequency of GnRH and LH pulses, and an increase in GnRH pulse size. No significant seasonal change was observed in the characteristics of GnRH and LH pulses in ovariectomized ewes in the absence of estradiol treatment. These findings lead to the conclusion that there is a marked seasonal change in the negative feedback effect of estradiol on episodic GnRH secretion in the ewe, with the steroid being maximally effective during anestrus.

Characterization and regulation of pre-ovulatory secretion of gonadotrophin-releasing hormone.

Using an elegant method for sampling of pituitary portal blood the secretory characteristics of gonadotrophin-releasing hormone (GnRH) during the oestradiol induced surge are studied. It is demonstrated that the neuroendocrine signal for ovulation in the ewe is a surge of GnRH released into the portal blood.

Distribution of estrogen receptor-immunoreactive cells in the sheep brain.

The distribution of immunoreactive (IR) estrogen receptor (ER)-containing cells was studied in the brains of adult Suffolk ewes using a rat monoclonal antibody (H222) which recognizes the human estrogen receptor. IR cells were characterized by dense nuclear reaction product, and in some instances, cytoplasmic immunostaining which filled dendrite-like processes. The greatest densities of ER-IR cells were found in the medial preoptic area, the mediobasal hypothalamus, and in a number of limbic system structures (amygdala, bed nucleus of the stria terminalis, lateral septum). ER-IR cells were found at lower densities in several other subregions of the hypothalamus and limbic system, and in the periaqueductal gray of the caudal midbrain. Cytoplasmic ER immunoreactivity was most prominent among ER-IR cells in the ventrolateral-ventromedial nucleus, the bed nucleus of the stria terminalis, the midbrain periaqueductal gray, and some ER-IR cells in the substantia innominata. The distribution of ER-containing cells in the sheep brain closely parallels that seen in other mammals. ER-IR cells are found in sites such as the medial preoptic area and ventrolateral-ventromedial hypothalamus which have been implicated as targets in this species and others for the influence of estradiol on sexual behavior and reproductive neuroendocrine function.

Fos expression during the estradiol-induced gonadotropin-releasing hormone (GnRH) surge of the ewe: induction in GnRH and other neurons.

The protein product of the protooncogene c-fos was used as a marker of cellular activation in an attempt to identify those neurons in the preoptic area and hypothalamus that participate in generation of the estradiol-induced surge of GnRH in the ewe. GnRH- and Fos-expressing cells were identified immunocytochemically, and the percent of coexpression was determined in three states: mid-luteal phase (low GnRH release, n = 6); short-term ovariectomy (high episodic GnRH release, n = 6); and induced GnRH surge (high sustained release, n = 8). To induce the GnRH surge, a follicular phase rise in circulating estradiol was simulated in a physiological model for the estrous cycle. Serum LH was measured as an indicator of GnRH release. In the luteal phase, LH was basal, indicating low GnRH secretion. Few cells expressed Fos; these were not GnRH cells. Despite high intermittent GnRH release in short-term ovariectomized ewes, GnRH cells did not express Fos. During the surge (sustained high GnRH release), 41 +/- 8% of GnRH cells expressed Fos; these cells were dispersed throughout the field of distribution of GnRH neurons. In addition to Fos in GnRH-positive cells, many more non-GnRH cells in the preoptic area, anterior hypothalamus, and ventrolateral hypothalamus expressed Fos during the surge than in the luteal phase or after ovariectomy. We suggest that Fos expression in GnRH cells is markedly increased by the positive feedback action of estradiol (surge), whereas short-term removal of negative feedback (ovariectomy) has little, if any, effect, despite increased GnRH release in both states. Since estradiol induces Fos expression in far more than GnRH neurons, our results also suggest that estradiol activates other cells, some of which may be part of a neuronal chain leading to GnRH surge generation, and some of which may be related to other neural actions of estradiol, such as estrous behavior.

Progesterone blocks the estradiol-induced gonadotropin discharge in the ewe by inhibiting the surge of gonadotropin-releasing hormone.

Previous studies indicate an elevation of circulating progesterone blocks the positive feedback effect of a rise in circulating estradiol. This explains the absence of gonadotropin surges in the luteal phase of the menstrual or estrous cycle despite occasional rises in circulating estradiol to a concentration sufficient for surge induction. Recent studies demonstrate estradiol initiates the LH surge in sheep by inducing a large surge of GnRH secretion, measurable in the hypophyseal portal vasculature. We tested the hypothesis that progesterone blocks the estradiol-induced surge of LH and FSH in sheep by preventing this GnRH surge. Adult Suffolk ewes were ovariectomized, treated with Silastic implants to produce and maintain midluteal phase concentrations of circulating estradiol and progesterone, and an apparatus was surgically installed for sampling of pituitary portal blood. One week later the ewes were allocated to two groups: a surge-induction group (n = 5) in which the progesterone implants were removed to simulate luteolysis, and a surge-block group (n = 5) subjected to a sham implant removal such that the elevation in progesterone was maintained. Sixteen hours after progesterone-implant removal (or sham removal), all animals were treated with additional estradiol implants to produce a rise in circulating estradiol as seen in the follicular phase of the estrous cycle. Hourly samples of pituitary portal and jugular blood were obtained for 24 h, spanning the time of the expected hormone surges, after which an iv bolus of GnRH was injected to test for pituitary responsiveness to the releasing hormone. All animals in the surge-induction group exhibited vigorous surges of GnRH, LH, and FSH, but failed to show a rise in gonadotropin secretion in response to the GnRH challenge given within hours of termination of the gonadotropin surges. The surges of GnRH, LH, and FSH were blocked in all animals in which elevated levels of progesterone were maintained. These animals in the surge-block group, however, did secrete LH in response to the GnRH challenge. We conclude progesterone blocks the estradiol-induced gonadotropin discharge in the ewe by acting centrally to inhibit the surge of GnRH secreted into the hypophyseal portal vasculature.

Seasonal changes of gonadotropin-releasing hormone secretion in the ewe.

A sustained volley of high-frequency pulses of GnRH secretion is a fundamental step in the sequence of neuroendocrine events leading to ovulation during the breeding season of sheep. In the present study, the pattern of GnRH secretion into pituitary portal blood was examined in ewes during both the breeding and anestrous seasons, with a focus on determining whether the absence of ovulation during the nonbreeding season is associated with the lack of a sustained increase in pulsatile GnRH release. During the breeding season, separate groups (n = 5) of ovary-intact ewes were sampled during the midluteal phase of the estrous cycle and following the withdrawal of progesterone (removal of progesterone implants) to synchronize onset of the follicular phase. During the nonbreeding season, another two groups (n = 5) were sampled either in the absence of hormonal treatments or following withdrawal of progesterone. Pituitary portal and jugular blood for measurement of GnRH and LH, respectively, were sampled every 10 min for 6 h during the breeding season or for 12 h in anestrus. During the breeding season, mean frequency of episodic GnRH release was 1.4 pulses/6 h in luteal-phase ewes; frequency increased to 7.8 pulses/6 h during the follicular phase (following progesterone withdrawal). In marked contrast, GnRH pulse frequency was low (mean less than 1 pulse/6 h) in both groups of anestrous ewes (untreated and following progesterone withdrawal), but GnRH pulse amplitude exceeded that in both luteal and follicular phases of the estrous cycle.(ABSTRACT TRUNCATED AT 250 WORDS)

Dynamics of gonadotropin-releasing hormone (GnRH) secretion during the GnRH surge: insights into the mechanism of GnRH surge induction.

Recent studies demonstrate unequivocally that a preovulatory surge of GnRH is secreted into pituitary portal blood during the estrous cycle of the ewe and that this surge is induced by the follicular phase rise in estradiol. These data, obtained at 10-min intervals, suggested the surge results from a continuous elevation of GnRH rather than from a sequence of discrete pulses. This study examines the dynamics of GnRH secretion in more detail to determine if the surge results from strictly episodic release of the decapeptide. Our approach was to monitor GnRH secretion into pituitary portal blood at very frequent intervals during several "windows" of the GnRH surge induced using a physiological model for the estrous cycle. Samples of portal blood were obtained at either 2-min intervals (6 ewes), or 30-sec intervals (12 ewes) at several times during the surge; at other times portal blood was sampled less often to monitor progression of the GnRH surge. All ewes had an unambiguous GnRH surge; amplitude ranged from 100- to 500-fold over pressure levels. Regardless of sampling interval, our results provide no convincing evidence to indicate the enhanced secretion of GnRH is strictly episodic; values remained continuously elevated in portal blood. Our findings are consistent with the hypothesis that the GnRH surge is not composed entirely of discrete synchronous secretory events, and they raise the possibility that one action of estradiol in inducing the GnRH surge may be to switch the pattern of GnRH secretion into portal blood from episodic to continuous.

Dynamics of gonadotropin-releasing hormone release during a pulse.

This study examined the nature of the GnRH signal that travels down the pituitary portal vessels and causes an LH pulse. Individual GnRH pulses were described in terms of abruptness of increase and decrease, amplitude, duration, and amount of GnRH released. Pituitary portal blood was obtained at 30-sec intervals for 2.5 or 5 h from five short-term ovariectomized ewes. Jugular blood was sampled every 10 min for LH. We examined 13 GnRH pulss; each produced an LH pulse. The contour of most GnRH pulses approximated a square wave. The rising edge of the GnRH pulse was very abrupt; GnRH secretion increased as much as 50-fold within 1 min. The mean peak amount of GnRH collected during pulses (24 pg/min, range 2-66) was 70-fold greater than the interpulse baseline (0.2-0.5 pg/min). The release period was sustained an average of 5.5 min; thereafter, GnRH fell to prepulse levels within 3 min. Overall, the larger and more prolonged pulses of GnRH were associated with higher amplitude LH pulses. To assess the distortion of the GnRH signal by the collection procedure, samples were obtained in vitro using the same technique during application of 4- and 7-min square wave GnRH pulses by means of a syringe pump. Signals were carried as square-waves through the sampling operation with minimal distoration, with the exception that amplitude decreased during the collection procedure. Our findings indicate the square-wave pulses observed in vivo are an accurate description of the dynamics of GnRH release during a pulse in short-term overiectomized ewes.