Dynamics of steroid regulation of GnRH secretion during the oestrus cycle of the ewe.

The oestrus cycle of the ewe is characterised by a long luteal phase followed by a short follicular phase and these periods are related to the production by the ovary of two major steroids: progesterone and oestrogen. Progesterone exerts a strong inhibitory effect on GnRH secretion during the luteal phase by a mechanism which is still unknown. Using an oestrogen-free ovine model and the portal blood collection technique we have obtained new insights into this mechanism. While progesterone removal induces a rapid increase in GnRH pulse frequency, progesterone reinsertion inhibits GnRH release even faster: less than 50 minutes. This action of progesterone is specific to the gonadotrophic axis and is mediated through an action on the nuclear receptor. Interestingly, this rapid mechanism is also strongly dependent of prior exposure to both progesterone and oestradiol. During the follicular phase, the rise in circulating oestradiol induces a robust preovulatory GnRH surge. In the ewe, this positive feedback effect is mainly exerted by an action of oestradiol on the mediobasal hypothalamus. Finally, we have also obtained evidence that progesterone priming is important for the full expression of the positive feedback action of oestradiol on GnRH secretion. In summary, progesterone and oestradiol sequentially exert opposite feedback effects on GnRH secretion during the oestrus cycle of the ewe but there is also clear evidence that the systems affected by these steroids are intimately linked.     

Optogenetic activation of GnRH neurons reveals minimal requirements for pulsatile luteinizing hormone secretion

The mechanisms responsible for generating the pulsatile release of gonadotropins from the pituitary gland are unknown. We develop here a methodology in mice for controlling the activity of the gonadotropin-releasing hormone (GnRH) neurons in vivo to establish the minimal parameters of activation required to evoke a pulse of luteinizing hormone (LH) secretion. Injections of Cre-dependent channelrhodopsin (ChR2)-bearing adeno-associated virus into the median eminence of adult GnRH-Cre mice resulted in the selective expression of ChR2 in hypophysiotropic GnRH neurons. Acute brain slice experiments demonstrated that ChR2-expressing GnRH neurons could be driven to fire with high spike fidelity with blue-light stimulation frequencies up to 40 Hz for periods of seconds and up to 10 Hz for minutes. Anesthetized, ovariectomized mice had optical fibers implanted in the vicinity of GnRH neurons within the rostral preoptic area. Optogenetic activation of GnRH neurons for 30-s to 5-min time periods over a range of different frequencies revealed that 10 Hz stimulation for 2 min was the minimum required to generate a pulse-like increment of LH. The same result was found for optical activation of GnRH projections in the median eminence. Increases in LH secretion were compared with endogenous LH pulse parameters measured from ovariectomized mice. Driving GnRH neurons to exhibit simultaneous burst firing was ineffective at altering LH secretion. These observations provide an insight into how GnRH neurons generate pulsatile LH secretion in vivo.Reproductive functioning in all mammals is critically dependent upon pulsatile gonadotropin secretion (1). Experiments undertaken in the 1980s clearly established that pulsatile luteinizing hormone (LH) and follicle-stimulating hormone secretion were generated by the episodic release of gonadotropin-releasing hormone (GnRH) into the pituitary portal vasculature (2–6). However, a quarter of a century since those experiments were performed, the components and mechanisms responsible for this episodic release of GnRH remain unknown and represent one of the most important unanswered questions in reproductive biology (7).Key parameters such as the number of GnRH neurons involved in a pulse and their patterns of electrical firing are unknown. An important insight into the dynamics of a GnRH pulse has come from fast portal blood sampling in ovariectomized sheep where each GnRH pulse is reported to approximate a square wave beginning sharply over 2 min, remaining elevated for ∼5 min, and then falling to baseline over the next 3 min (8). This allowed speculation that a subgroup of GnRH neurons may fire coordinately for a period of 2–7 min to generate a pulse of GnRH (7). Disappointingly, however, direct electrical recordings of adult GnRH neurons in acute brain slices in vitro have provided no clear correlate of pulsatile hormone secretion (7, 9). Recent investigations into GnRH neuron firing in vivo in anesthetized GnRH-green fluorescent protein (GFP) mice have similarly been unable to shed light on the pulse-generating properties of these cells (10). The most promising insights into the nature of GnRH pulsatility have come from studies of embryonic GnRH neurons in vitro where episodes of burst firing, represented by calcium transients, are found to synchronize occasionally in subpopulations of GnRH neurons in a time frame similar to that of pulsatile GnRH/LH secretion (11, 12).The best way of determining the patterns of GnRH neuron firing that generate an LH pulse would be to record the activity of hypophysiotropic GnRH neurons while simultaneously measuring LH secretion in vivo. At present this remains impossible. An alternative approach that might shed light on this issue would be to determine the minimal patterns of GnRH neuron firing that are capable of generating an LH pulse in vivo. This is now possible using optogenetic approaches, and we report here a strategy that allows hypophysiotropic GnRH neurons to be transfected with channelrhodopsins (ChR2) and subsequently activated in vivo to generate pulses of LH secretion. This reveals that GnRH neurons need only be activated at either their cell bodies or distal projections within the median eminence (ME) for 2 min at a constant 10-Hz firing rate to generate an LH pulse. Surprisingly, synchronizing burst firing among GnRH neurons is ineffective.     

The pattern of ovarian inhibin, estradiol, and androstenedione secretion during the estrous cycle of the ewe

An experiment was conducted in order to determine the pattern of, and the relationships between, the secretion of inhibin, estradiol, and androstenedione by the ovary and the concentration of LH, FSH, and PRL during the estrous cycle of sheep. The estrous cycles of 6 Finn-Merino ewes in which the left ovary had been autotransplanted to the neck were synchronized by two injections of cloprostenol (100 micrograms im) a potent analog of prostaglandin F2 alpha (PG) given 14 days apart. The ewes had ovarian and jugular venous blood samples taken at four hourly intervals from 42 h before the second PG injection until day 6 of the following cycle. All animals responded to PG with the preovulatory LH surge occurring within 58 +/- 2 h (mean +/- SEM). The concentration of FSH in jugular venous plasma fell (P less than 0.001) after the induction of luteolysis and then exhibited 3 peaks, the first coincident with the LH surge, the second on day 1, and the third on day 6. After injection of PG the secretion rates of inhibin, estradiol, and androstenedione increased (P less than 0.05) within 4-8 h. After this increase in the early follicular phase the secretion rate of estradiol continued to rise until the time of the LH surge (P less than 0.001). Although the secretion of androstenedione and inhibin increased in the 36 h before the LH surge the magnitude of this rise was less marked than for estradiol and was not statistically significant. Within 4-8 h of the start of the LH surge the secretion of estradiol and androstenedione declined rapidly reaching barely detectable levels within 16 h (P less than 0.001). In contrast the secretion of inhibin increased after the LH surge reaching a broad peak (P less than 0.05) of approximately 16-h duration, coincident with the second peak of FSH. From days 2-6 mean secretion of inhibin remained relatively stable at 2-6 ng/min although considerable variation was observed in individual profiles. The rate of estradiol secretion increased steadily from its nadir on day 1 to a broad peak centered around day 3 (3-6 ng/min, P less than 0.001) followed by a decline until by day 6 the estradiol secretion rate was less than 1 ng/min (P less than 0.01). The secretory profile for PRL showed a close relationship with estradiol secretion.(ABSTRACT TRUNCATED AT 400 WORDS)     

Characteristics of the plasma melatonin rhythm are not modified by steroids during the estrous cycle in Ile-de-France ewes

Abstract: Two experiments were designed to determine whether gonadal steroids during the estrous cycle may modify the characteristics of the plasma melatonin rhythm. In the first experiment, 12 ovariectomized estradiol-treated ewes were used and exposed to constant short days. The experimental design was a latin square to distinguish between steroid treatments and individual effects on melatonin secretion. Twenty four hours before the bleeding period (hourly during 20 hr) and with a 1 week interval, animals were treated with a) additional subcutaneous estradiol implants, b) progesterone devices, or c) control. In the second experiment, nine ewes received a treatment combining fluorogestone acetate devices and pregnant mare serum gonadotrophin to induce synchronous ovulations. Samples for melatonin determination were obtained hourly for 13 hr at three stages of estrous cycle: follicular phase, early luteal phase, and late luteal phase. Ovarian activity was monitored by taking daily samples for progesterone analysis and ovulation rate was determined by laparoscopy. Duration and mean melatonin plasma concentrations of the elevation were calculated for each ewe and each night and analysed by latin square test (experiment 1) or ANOVA (experiment 2). Melatonin concentrations of elevation and duration of elevation were not significantly affected by hormonal treatments or by phase of estrous cycle. A strong individual effect was detected ( P <0.01) for both parameters in both experiments. It was concluded that melatonin secretion is unaffected by steroid administration or by phase of estrous cycle. The existence of very high inter-individual variation suggest that both parameters are individual characteristics of each animal which may have a strong genetic basis.     

In vivo imaging of the GnRH pulse generator reveals a temporal order of neuronal activation and synchronization during each pulse

A hypothalamic pulse generator located in the arcuate nucleus controls episodic release of gonadotropin-releasing hormone (GnRH) and luteinizing hormone (LH) and is essential for reproduction. Recent evidence suggests this generator is composed of arcuate “KNDy” cells, the abbreviation based on coexpression of kisspeptin, neurokinin B, and dynorphin. However, direct visual evidence of KNDy neuron activity at a single-cell level during a pulse is lacking. Here, we use in vivo calcium imaging in freely moving female mice to show that individual KNDy neurons are synchronously activated in an episodic manner, and these synchronized episodes always precede LH pulses. Furthermore, synchronization among KNDy cells occurs in a temporal order, with some subsets of KNDy cells serving as “leaders” and others as “followers” during each synchronized episode. These results reveal an unsuspected temporal organization of activation and synchronization within the GnRH pulse generator, suggesting that different subsets of KNDy neurons are activated at pulse onset than afterward during maintenance and eventual termination of each pulse. Further studies to distinguish KNDy “leader” from “follower” cells is likely to have important clinical significance, since regulation of pulsatile GnRH secretion is essential for normal reproduction and disrupted in pathological conditions such as polycystic ovary syndrome and hypothalamic amenorrhea.

Reproduction in mammals depends on a hypothalamic pulse generator that regulates the episodic release of gonadotropin-releasing hormone (GnRH) from the hypothalamus (1, 2). Regulation of the frequency and amplitude of GnRH pulses, and, in turn, that of the gonadotropins luteinizing hormone (LH) and follicle-stimulating hormone from the anterior pituitary gland, is essential for steroid hormone production and gamete development at the gonads. Although the first observations of the pulsatile nature of GnRH and LH release were made in the 1970s, until recently the precise location and cellular identity of the neural pulse generator responsible for episodic GnRH release remained a major unanswered question.The 2003 discovery that mutations in the gene encoding the kisspeptin receptor (G protein-coupled protein 54 [GPR54]) result in hypogonadotropic hypogonadism delivered compelling evidence that kisspeptin-positive cells in the brain are required for maintaining GnRH release (3, 4). Subsequent studies testing the role of kisspeptin in animal models confirmed activation of GnRH neurons via GPR54 to potently stimulate GnRH and LH release (5, 6). Multilabeling experiments in sheep later revealed that kisspeptin cells in the arcuate nucleus of the hypothalamus (ARC) also coexpressed two other important mediators of GnRH release, the tachykinin neurokinin B (NKB) and the endogenous opioid peptide dynorphin (7); as an abbreviation, these cells were termed KNDy (kisspeptin/neurokinin B/dynorphin) neurons. Colocalization of KNDy peptides was subsequently demonstrated in the mouse, rat, cow, goat, and nonhuman primate (8–12). Anatomical characterization of KNDy neurons revealed reciprocal connections and the expression of postsynaptic receptors for NKB and dynorphin, indicating KNDy cells form an interconnected population potentially capable of synchronization (9, 13–16). These characteristics provided the basis for the “KNDy hypothesis” of GnRH pulse generation, in which NKB acts as the signal responsible for pulse onset by triggering activation of reciprocally connected KNDy neurons and driving the kisspeptin-mediated secretion of GnRH (13, 17–20). In support of this, bilateral infusions of NKB or dynorphin antagonists into the mediobasal hypothalamus enhances and suppresses LH pulsatile release, respectively, in sheep and goats (12, 21). Further, the conditional inhibition and brief activation of kisspeptin-expressing cells in the ARC using optogenetic tools in mice suppresses and elicits LH pulses, respectively (22, 23). Finally, in vivo measurement of KNDy neuron population activity using GCaMP6 fiber photometry in awake and freely moving mice revealed transient increases in intracellular calcium by KNDy neurons before an LH pulse, indicative of episodic activity within the KNDy neuron population that drives pulsatile LH release (22). However, as these studies either manipulate or record the activity of large proportions of the KNDy population, they do not identify individual cells which are responsible for GnRH/LH pulse generation, or whether those cells are activated homogeneously during an individual pulse. To address these questions, we conducted in vivo calcium imaging using miniature microscopy in order to visualize KNDy cell activity at the single-cell level in freely behaving mice, and, combined this with serial blood sampling, to examine the pattern of activation of KNDy cells during an LH pulse. Using this imaging approach, we provide in vivo evidence that directly demonstrates the synchronization among individual KNDy cells that was first theorized by the KNDy hypothesis and demonstrate that synchronized activation always precedes an individual LH pulse. Unexpectedly, we also found that not all KNDy cells are activated simultaneously during a pulse; rather, KNDy cells are recruited in a recurring temporal order composed of distinct subpopulations of “leader” cells, which activate and reach peak activity at the onset of synchronized episodes, and “follower” cells, which reach peak activity during either the maintenance or termination phase of the episode. These results provide a major advance to our understanding of oscillatory neuroendocrine systems by demonstrating that LH pulse secretion is preceded by highly synchronized activity among individual KNDy cells in an in vivo setting. In addition, the existence of temporally defined subpopulations of “leader” and “follower” cells in each synchronized event is strikingly similar to results described recently in pancreatic beta cells (24), suggesting that this temporal organization may be a common feature of endocrine pulse generators in the brain as well as in the periphery.     

Pulsatile leptin secretion is independent of luteinizing hormone secretion in prepubertal sheep

Many studies have suggested that leptin modulates the gonadal axis. A synchronicity of luteinizing hormone (LH) and leptin has been described in humans, suggesting that leptin may modulate the episodic secretion of LH. The objective of this study was to establish whether episodic leptin secretion depends on the episodic LH secretion in prepubertal sheep. We used two different approaches. The first consisted of blocking the release of LH using a long-acting LH-releasing hormone (LHRH) agonist and analyzing the episodic LH and leptin secretions. The second method stimulated the pituitary gland with pulses of LHRH and again LH and leptin secretions were analyzed. Spring-born 20-wk-old Suffolk ewe lambs (n = 5) received intramuscularly a long-acting LHRH agonist (Decapeptyl). Treatment was repeated at 24 and 28 wk of age. Control lambs (n = 6) received the vehicle of Decapeptyl. Diurnal and nocturnal pulsatilities of LH and leptin were studied at 20 (before Decapeptyl injection), 26, and 30 wk. Blood samples were taken at 10-min intervals for 6 h, beginning at 10:00 AM (diurnal sampling) and at 10:00 PM (nocturnal sampling). In all samples, LH and leptin were measured by radioimmunoassay, and pulsatile hormone secretion characteristics were assessed by the CLUSTER program. To characterize further the synchronicity between LH and leptin pulses, LHRH (10 ng/kg body wt) was injected at 60-min intervals, six times, to another five 30-wk-old ewe lambs, for the same time period as the pulsatility study. In the control group, LH secretion did not change between lambs of 20 and 30 wk of age. In LHRH agonist-treated lambs, LH secretion diminished from 20 to 30 wk of age and was lower than in control lambs at 26 and 30 wk of age (p < 0.05). The transversal mean (ng/[mL x 6 h]) of leptin concentrations was different between control lambs of 20 wk of age and 26 and 30 wk of age (p < 0.01). Contrary to the findings in LH secretion, in LHRH agonist-treated lambs, mean plasma leptin concentrations did not decrease. Furthermore, the mean diurnal and nocturnal leptin concentrations and the pulse amplitude were higher at 26 and 30 wk than at 20 wk in LHRH agonist-treated lambs (p < 0.05). There were no differences between diurnal and nocturnal parameters of leptin secretion in both groups. There was no synchronicity between LH and leptin pulses. LHRH pulses significantly increased plasma LH concentrations, producing discernible LH pulses; however, leptin amplitude and leptin pulse frequency were not modified by the exogenous LHRH pulses, exhibiting no coincidence with LH pulses. The data suggest that pulsatile leptin secretion is independent of LH secretion in ewe lambs.     

Pituitary content of gonadotropins and receptors for gonadotropin-releasing hormone (GnRH) and hypothalamic content of GnRH during the periovulatory period of the ewe

Studies were undertaken to determine if the number of hypophyseal receptors for GnRH changes at the time of the preovulatory surge of LH in ewes. Concentrations of LH, FSH, progesterone, and estradiol in serum and concentrations of LH and FSH in pituitary were measured. The content of GnRH in the hypothalamus was also determined. Estrus was synchronized in 35 cross-bred ewes by injecting prostaglandin F2 alpha (PGF2 alpha) at 0 and 4 h (7.5 mg each, im) on day 14 of a naturally occurring estrous cycle, followed 30 h later by the injection of estradiol (25 micrograms in safflower oil, im). Five ewes were killed at each of the following times relative to the first injection of PGF2 alpha: 0, 24, 32, 44, 50, 56 and 96 h. Blood samples were collected throughout the course of the experiment. Concentrations of progesterone in serum decreased markedly by 8 h after PGF2 alpha and were uniformly undetectable (less than 300 pg/ml) by 34 h. Concentrations of estradiol in serum increased after the injection of estradiol and returned to basal values 10 h later. Surges of LH, which were usually coincident with surges of FSH, occurred between 43 and 53 h. Concentrations of both LH and FSH in the pituitary declined after the LH surge. There were no significant changes in the amount of GnRH contained in the preoptic area, the median eminence, or the hypothalamus. The number of receptors for GnRH increased at 24 and 32 h compared to the 0 h value and remained elevated at 44 and 50 h. After the LH surge (56 h), the number of GnRH receptors declined and at 96 h was not different from the number measured at 0 h. Since an increase in the number of receptors will result in the formation of more receptor-hormone complex and may lead to an augmented response, these data suggest that an increase in the number of hypophyseal receptors for GnRH may contribute to the preovulatory LH surge in ewes.     

Changes in the pulsatile pattern of luteinizing hormone secretion during the rat estrous cycle

To ascertain whether changes in the pattern of GnRH release from the hypothalmus occur during the 4-day rat estrous cycle, the pattern of LH release was characterized on each day of the estrous cycle, and the results were interpreted in light of the changes in pituitary responsiveness to GnRH previously described by this laboratory to occur during this time. Blood samples were taken from intact, freely moving rats via venous catheters at 6- to 10-min intervals for 3-4 h. LH pulse height and LH interpulse interval were quantified on each day of the cycle, and the transition on the afternoon of proestrus from tonic LH release to the preovulatory LH surge was detailed. The effects on the pattern of LH release during estrus of small doses of GnRH (0.4 ng) and the continuous infusion of the opioid antagonist naloxone were also examined. Plasma LH concentrations (NIAMDD rat LH-RP-1) were determined with a highly sensitive LH RIA. LH pulses were identified using the PULSAR algorithim. The LH interpulse intervals of 46 +/- 2 min on diestrous-1 day, 49 +/- 4 min on diestrous day 2, and 60 +/- 8 min on proestrus immediately before the LH surge were not significantly different. There were no changes immediately preceding the preovulatory LH surge on the afternoon of proestrus in either the LH interpulse interval or the LH pulse height. Instead, the transition from tonic LH secretion to the preovulatory LH surge was found to occur abruptly. These data suggest that an abrupt increase in GnRH secretion during the afternoon of proestrus initiates the dramatic rise in LH concentrations. The pattern of LH secretion during the day of estrus differed significantly from that on the other days of the cycle in that no LH pulses were observed. However, the administration of small pulses of GnRH elicited physiological elevations in LH release. Furthermore, the continuous infusion of naloxone to estrous rats immediately stimulated a pulsatile pattern of LH secretion, with a LH interpulse of 56 +/- 4 min. These data indicate that the absence of LH pulses during estrus may result from a deficit in GnRH release. Similar modifications in GnRH release during the other days of the cycle were inferred from the observed changes in LH pulse heights. The LH pulse height of 21 +/- 3 ng/ml on diestrous day 2 was significantly less than the LH pulse height of 41 +/- 4 ng/ml on diestrous day 1 or 35 +/- 4 ng/ml on proestrus before the surge.(ABSTRACT TRUNCATED AT 400 WORDS)     

Melatonin secretion decreases during the proestrous stage of the rat estrous cycle.

Urine was collected from rats during 12 consecutive daily dark periods and assayed for melatonin and norepinephrine; the phase of the vaginal estrous cycle associated with each urine sample was determined from daily vaginal smears. The proestrous phase of the estrous cycle was consistently associated with significant reductions in the excretions of both compounds. The level of melatonin in any urine sample tended to vary as a function of its norepinephrine content; however, the slope of the curve relating these two compounds in metestrous-diestrous samples differed from that for proestrous-estrous specimens. This difference suggests that factors other than the catecholamine (e.g., gonadal hormones) also affect melatonin secretion. Oophorectomy elevated the melatonin concentration of serum but not that of the pineal; this rise was suppressed by the administration of estrogen plus progesterone. The fate of circulating melatonin (as indicated by the proportion of an exogenous dose excreted into the urine) was not affected by the state of the estrous cycle.     

Autocrine regulation of prolactin secretion by endothelins throughout the estrous cycle

We have previously found that the ovarian steroid background determines the efficiency of the endothelin-mediated autocrine feedback regulation of prolactin (PRL) secretion. In this study, we investigated the role of endogenous endothelins in regulating PRL secretion during the estrous cycle. Adult female rats representing different stages of the 4-d cycle were sacrificed by decapitation, and the anterior pituitary cells were enzymatically dispersed using collagenase and hyaluronidase. PRL secretion of individual lactotrophs was measured in a PRL-specific reverse hemolytic plaque assay, and the influence of endogenous endothelins on PRL secretion was assessed by applying the selective ET_A receptor antagonist peptide, BQ123. Blocking the endothelin-mediated autocrine feedback resulted in an increase in PRL secretion when cells were obtained at proestrus, estrus, and diestrus-1, whereas PRL secretion was decreased at diestrus-2 by ET_A receptor blockade. These observations suggest that endogenous endothelins are predominantly inhibitory during proestrus, estrus, and diestrus-1, whereas at diestrus-2 their influence on PRL secretion in stimulatory. Whereas the bell-shaped concentration-response curves with BQ123 at proestrus and diestrus-1 may indicate a transition state in which endogenous endothelins can be both stimulatory and inhibitory, at estrus the influence of endogenous endothelins is unequivocally inhibitory in nature. We propose that intensification of the endogenous endothelin-mediated negative feedback at estrus may play a role in restraining PRL secretion following the estradiol-induced proestrous PRL surge. This article is dedicated to the memory of L. Stephen Frawley.