Presynaptic inhibition decreases when estrogen level rises.

The objective was to determine estrogen's influence on control of a skeletal muscle through measurements of motorneuron excitability (H:M ratio) and presynaptic inhibition (PI). Estrogen serum concentrations were measured at menses and ovulation of female subjects and compared to male controls. Data were analyzed from 12 women and 13 men reporting no history of knee ligament injury. Women reported regular menstrual cycles and no hormone-based contraceptive use for the previous year. Women were tested at menses (Time1) and ovulation (Time2). Men were tested twice, approximately 14 days apart. Analysis indicated no difference in the H:M ratio between the sexes at either time point. A significant difference for the sexes was detected in the magnitude of estrogen change (∆EST) between observations. At Time1, the male and female estrogen concentrations were not different; however, they were different at Time2, primarily due to the large rise observed in the women. A significant difference between the sexes was also seen in the magnitude of change for PI (∆PI) between observations. As with EST, the levels of PI between the sexes at Time1 were not different; however, a difference existed at Time 2. Estrogen interacts with GABA at several nervous system locations affecting inhibition of synaptic transmission. This is the first study to investigate changes in PI of a skeletal muscle between times of low and high estrogen. Improving the understanding of estrogen's influence on skeletal muscles may provide answers to why noncontact anterior cruciate ligament injuries of the knee occur more frequently in women.

Antagonism of serotonin receptor 1B decreases viability and promotes apoptosis in the COS canine osteosarcoma cell line.

Serotonin receptor 1B (5HTR1B) traditionally exhibits anti-proliferative activity in osteoblasts. We examined the expression and function of 5HTR1B in the COS canine osteosarcoma cell line and normal canine osteoblasts. Equal levels of 5HTR1B gene and protein expression were found between normal and malignant osteoblasts. Treatment with serotonin enhanced viability of osteosarcoma cells but not normal osteoblasts. Challenge with the 5HTR1B agonist anpirtoline caused no change in cell viability. Rather incubation with the specific receptor antagonist SB224289 caused reduction in osteoblast viability, with this effect more substantial in osteosarcoma cells. Investigation of this inhibitory activity showed 5HTR1B antagonism induces apoptosis in malignant cells. Evaluation of phosphorylated levels of CREB and ERK, transcriptional regulators associated with serotonin receptor signalling in osteoblasts, revealed aberrant 5HTR1B signalling in COS. Our results confirm the presence of 5HTR1B in a canine osteosarcoma cell line and highlight this receptor as a possible novel therapeutic target.

Oestrogen induces rhythmic expression of the Kisspeptin-1 receptor GPR54 in hypothalamic gonadotrophin-releasing hormone-secreting GT1-7 cells.

Oestrogen-stimulated preovulatory gonadotrophin surges are temporally regulated in a way that remains not fully understood. Mammalian ovulation requires surges of gonadotrophin-releasing hormone (GnRH), released from specialised neurones in the hypothalamus. Surge regulation is mediated by ovarian oestrogen (17 ß-oestradiol; E(2) ) feedback-acting as a negative signal until the early afternoon of the pro-oestrous phase, at which point it stimulates robust increases in GnRH release. Multiple lines of evidence suggest a role for the circadian clock in surge generation, although the presence of endogenous oscillators in several neuronal populations throughout the mediobasal hypothalamus complicates an elucidation of the underlying mechanisms of circadian regulation. In the present study, we propose that endogenous oscillators within GnRH neurones are modulated by oestrogen to elicit GnRH surge secretion. One mechanism by which this may occur is through the up-regulation of receptors of known stimulators of GnRH, such as kisspeptin's cognate receptor, GPR54. Through analysis of mRNA and protein abundance patterns, we found that high levels of E(2) elicit circadian expression profiles of GPR54 in vitro, and that disruption of endogenous GnRH oscillators of the clock dampens this effect. Additionally, although kisspeptin administration to GT1-7 cells does not result in surge-level secretion, we observed increased GnRH secretion from GT1-7 cells treated with positive feedback levels of E(2) . These results in this in vitro neuronal model system suggest a possible mechanism whereby receptor expression levels, and thus GnRH sensitivity to kisspeptin, may change dramatically over the pro-oestrous day. In this way, elevated ovarian E(2) may increase kisspeptidergic tone at the same time as increasing GnRH neuronal sensitivity to this neuropeptide for maximal surge release.

Modulation of gonadotrophin-releasing hormone secretion by an endogenous circadian clock.

The mechanisms mediating positive feedback effects of oestradiol on pre-ovulatory gonadotrophin releasing-hormone (GnRH) surge generation in female mammals, although well-explored, are still incompletely understood. In addition to binding to and signalling through classical nuclear receptor-mediated pathways in afferent hypothalamic neurones, recent evidence suggests that ovarian steroids may use membrane-bound receptors or nonclassical signalling pathways to directly influence cell function leading to the generation of GnRH surge secretion. We review recent investigations into the role of the endogenous molecular circadian clock on modulation of GnRH gene expression and neuropeptide secretion, and will explore potential molecular mechanisms by which ovarian steroids may directly induce secretory changes at the level of the GnRH neurone, examining closely whether circadian clock gene oscillations may be involved.

Clocks and the black box: circadian influences on gonadotropin-releasing hormone secretion.

Although the mechanisms underlying hypothalamic surge secretion of gonadotropin-releasing hormone (GnRH) in rodent models have remained enduring mysteries in the field of neuroendocrinology, the identities of two fundamental constituents are clear. Elevated ovarian oestrogen, in conjunction with circadian signals, combine to elicit GnRH surges that are confined to the afternoon of the proestrus phase. The phenomenon of oestrogen positive feedback, although extensively investigated, is not completely understood, and may involve the actions of this steroid directly on GnRH perikarya, as well as on the activity of neuronal afferents. Additionally, whereas many studies have focused upon regulation of GnRH surge secretion by the neuroanatomical biological clock, the suprachiasmatic nucleus, it remains unclear why this daily signal is capable of stimulating surges only in the presence of oestrogen. This review re-examines multiple models of circadian control of reproductive neurosecretion, armed with the recent characterisation of the intracellular transcriptional feedback loops that comprise the circadian clock, and attempts to evaluate previous studies on this topic within the context of these new discoveries. Recent advances reveal the presence of oscillating circadian clocks throughout the central nervous system and periphery, including the anterior pituitary and hypothalamus, raising the possibility that synchrony between multiple cellular clocks may be involved in GnRH surge generation. Current studies are reviewed that demonstrate the necessity of functional clock oscillations in generating GnRH pulsatile secretion in vitro, suggesting that a GnRH-specific intracellular circadian clock may underlie GnRH surges as well. Multiple possible steroidal and neuronal contributions to GnRH surge generation are discussed, in addition to how these signals of disparate origin may be integrated at the cellular level to initiate this crucial reproductive event.

In vivo gonadotropin-releasing hormone secretion in female rats during peripubertal development and on proestrus.

Pubertal development in female rats is characterized by increased LH levels and the appearance of estrogen-dependent afternoon LH mini-surges. In these studies we performed the first analysis of GnRH patterns in peripubertal rats to determine whether there are similar changes in pulsatile GnRH release. Microdialysis samples were collected at 5-min intervals throughout a 5-h afternoon period from 22 rats sampled on a single day between 30-47 days of age. Adult female rats were sampled on proestrus for comparison. In 30- to 33-day-old rats, GnRH release was infrequent (2.7 pulses/5 h; n = 3), whereas intermediate pulse frequencies were observed in 34- to 37-day-old rats (6.4 pulses/5 h; n = 9) and 38- to 42-day-old (5.0 pulses/5 h; n = 5) rats. The highest GnRH pulse frequencies were observed in 43- to 47-day-old rats (9.4 pulses/5 h; n = 5). Mean GnRH pulse amplitude did not vary significantly with age. Animals sampled before vaginal opening (VO) exhibited significantly slower GnRH pulse frequencies than those sampled after vaginal opening (1.3 pulses/5 h pre-VO vs. 7.6 pulses/5 h post-VO; P = 0.01). An afternoon increase in GnRH secretion, defined operationally as a greater than 25% increase in mean GnRH levels in the last half of the sampling period and tentatively termed a mini-surge, was observed in 0%, 33%, 40%, and 60% of 30- to 33-, 34- to 37-, 38- to 42-, and 43- to 47-day-old rats, respectively. An overall increase in GnRH pulse frequency was observed in females displaying a mini-surge (9.0 pulses/5 h with mini-surge compared with 4.7 pulses/5 h with no mini-surge). The mini-surge itself, however, was associated with a late afternoon increase in GnRH pulse amplitude and not in pulse frequency. In adult proestrous rats, peak levels during the GnRH surge were an order of magnitude greater than those reached in pubertal animals. Our findings demonstrate that pubertal maturation in the female rat is associated with an acceleration of GnRH pulse generator activity and that later stages of pubertal maturation are characterized by the appearance of afternoon increases in GnRH release that may underlie previously reported mini-surges in LH.

Progesterone receptors as neuroendocrine integrators.

Intracellular progesterone receptors (PRs) are ligand-inducible transcription factors that mediate the majority of the effects of progesterone (P) on neuroendocrine functions. During the past decade, evidence has accumulated which suggest that PRs can also be activated independently of P, by signals propagated through membrane-bound receptors to the interior of cells. The activation of PRs by this type of "cross-talk" mechanism has been implicated in the physiological regulation of several important neuroendocrine processes, including estrous behavior and periovulatory hormone secretions. We review evidence that both ligand-dependent and ligand-independent activation of PRs occurs in central neurons and in anterior pituitary cells and that the convergence and summation of these signals at the PR serves to integrate neural and endocrine signals which direct several critically important neuroendocrine processes. An integrative function for PRs is reviewed in several physiological contexts, including the display of lordosis behavior in female rodents, the neurosecretion of gonadotropin-releasing hormone surges, secretion of preovulatory gonadotropin surges, and release of periovulatory follicle stimulating hormone surges. The weight of evidence indicates that cross talk at the intracellular PR is an essential component of the integrative mechanisms that direct each of these neuroendocrine events. The recurrence of PR's integrative actions in several different physiological contexts suggests that other intracellular steroid receptors similarly function as integrators of neural and endocrine signals in other neuroendocrine processes.

Stimulation of gonadotropin-releasing hormone surges by estrogen. I. Role of hypothalamic progesterone receptors.

Estrogen (E2) stimulates GnRH surges by coupling a daily neural signal to neuronal circuitries governing GnRH release. We have hypothesized that E2 promotes this coupling process by inducing expression of neuronal transcription factors, which are subsequently activated by neurotransmitter-mediated mechanisms representing the daily neural signal. These experiments tested the specific hypothesis that the progesterone receptor (PR) functions in this manner, viz. as an E2-induced factor whose activation is necessary for the stimulation of GnRH surges. Two complimentary experiments were performed to determine whether activation of hypothalamic PRs is obligatory for the stimulation of GnRH surges by E2. In the first, the effects of a PR antagonist on GnRH and LH surges were assessed in ovariectomized (OVX), E2-primed rats. Rats were OVX on diestrous day 2, treated with 30 microg estradiol benzoate or oil vehicle, sc, and then administered either oil vehicle or the type I antiprogestin, ZK98299 at 0900 h on proestrus. GnRH release rates and plasma LH levels were determined in each animal by microdialysis of median eminence and atrial blood sampling, respectively. Estrogen, but not oil vehicle, treatment evoked robust and contemporaneous GnRH and LH surges in animals that received no PR antagonist on proestrus. Additional treatment with ZK98299, however, completely blocked both GnRH and LH surges. In a second experiment, specific involvement of anteroventral periventricular (AVPV) PRs in E2-induced GnRH surges was assessed. Additional groups of OVX, E2-primed rats were fitted with intracerebroventricular cannulas, and PR antisense oligonucleotides were infused into the third ventricle adjacent to the AVPV to prevent expression of PR in this periventricular region. Animals infused with PR antisense oligos did not exhibit any LH surges, whereas surges were observed in saline-, missense-, and sense oligo-treated controls. Immunohistochemistry confirmed the effectiveness of PR antisense oligonucleotides in blocking PR expression. These findings provide direct support for the hypothesis that activation of PRs, specifically those in hypothalamic regions including the AVPV, is an obligatory event in the stimulation of GnRH surges by E2.

Stimulation of gonadotropin-releasing hormone surges by estrogen. II. Role of cyclic adenosine 3'5'-monophosphate.

Release of GnRH surges in female rats is directed by a daily neural signal and occurs only after exposure of the hypothalamus to sustained, elevated estrogen (E2) levels in serum. We have proposed that preovulatory E2 couples the daily neural signal to the circuitry governing GnRH release by a two-step process, which includes stimulation of neuronal progesterone receptors (PRs) by E2 and subsequent activation of PRs by the daily neural signal. In the preceding report we documented that PR activation is obligatory for the stimulation of GnRH surges by E2. In these studies we assess the validity of a second essential feature of this model, that neural signals can activate PRs and thereby prompt the release of GnRH and LH surges. Our efforts specifically focused on the role of cAMP in mediating neural PR trans-activation leading to GnRH surges. To assess whether cAMP may function as a daily neural signal, cAMP levels were examined via a competitive binding assay in anteroventral periventricular nucleus (AVPV) homogenates obtained at 0900, 1200, 1500, 1800, and 2100 h on all days of the estrous cycle. A significant rise in cAMP concentrations was observed at 1500 h on all estrous cycle days. A similar rise at the same time was observed in AVPV tissues of ovariectomized (OVX) rats regardless of steroid treatment. No significant increase in cAMP levels was observed at any time point in homogenates of ventromedial nucleus or cerebral cortex. In a second experiment, female rats were OVX on the afternoon of diestrous day 2 and simultaneously administered 30 microg estradiol benzoate or oil vehicle. On the following day of presumptive proestrus, rats received intracerebroventricular infusions of the cAMP analog, 8-bromo-cAMP, or saline vehicle at 0900 h. Rats treated with 8-bromo-cAMP exhibited LH surges that were advanced by 3 h compared with those in saline-treated controls. This advance did not occur in 8-bromo-cAMP-treated rats not primed with E2, or in E2-treated rats given the antiprogestin RU486. In a third experiment, OVX, estradiol benzoate-primed rats received intracerebroventricular infusions of saline vehicle or the adenylyl cyclase inhibitor SQ22536; although saline-treated rats exhibited normal LH surges, no surges were observed in the rats receiving SQ22536. In additional SQ22536-treated animals, however, LH surge release was rescued and greatly augmented by a pharmacological dose of progesterone. These results demonstrate that 1) cAMP levels in the AVPV are significantly elevated at 1500 h on a daily basis; 2) cAMP elevations in the AVPV can prematurely evoke LH surges by a mechanism that requires PR activation; 3) inhibition of adenylyl cyclase activity in the AVPV blocks LH surges, an action that can be reversed by progesterone; and 4) cAMP generation leads to PR transactivation in the AVPV. Our observations thus provide support for the hypothesis that an increase in intracellular cAMP in the AVPV acts as a component of the daily neural signal required to initiate GnRH and subsequent LH surges, and that transmission of this signal is mediated by cAMP-induced PR trans-activation in the AVPV.

Absence of gonadotropin surges and gonadotropin-releasing hormone self-priming in ovariectomized (OVX), estrogen (E2)-treated, progesterone receptor knockout (PRKO) mice.

It is well known that estrogen (E2) stimulates expression of progesterone receptors (PRs), thereby inducing responsiveness of several tissues to the actions of progesterone (P). Recent studies have also suggested, however, that biological actions previously ascribed to E2 alone may also be mediated by activation of E2-induced PRs, even independently of signal changes in P concentrations. In the present experiments, the progesterone receptor knockout (PRKO) mice were used to assess the role of PR activation in the positive feedback actions of E2 on gonadotropin release. Ovariectomized (OVX) PRKO mice were tested for their capacity to mount primary gonadotropin surges in response to exogenous E2, and to exhibit a GnRH self-priming effect in response to sequential injections of the decapeptide. Wild-type (WT) and PRKO mice were OVX, treated with both 17beta-estradiol and estradiol benzoate (EB), and then killed at 1900 h on day 7 postOVX. Plasma LH RIA revealed that WT mice exhibited surges in response to the E2 treatment; the PRKO mice, however, showed no elevation in plasma LH above untreated controls. Instead, plasma LH levels in E2-treated, OVX PRKO mice decreased significantly in comparison to untreated OVX PRKO mice, suggesting that E2 can exert a negative feedback influence on LH release in PRKO mice, despite the absence of positive feedback effects. A slight but significant rise in plasma FSH was observed in E2-treated OVX WT mice in comparison to untreated controls: an effect not seen in E2-treated OVX PRKO mice, reinforcing the observation that estrogen's positive feedback effects are compromised in PRKO mice. In a second experiment, E2-treated OVX WT and PRKO mice were given either one or two pulses of GnRH 60 min apart, and killed 10 min later. The WT mice were found to exhibit a robust GnRH self-priming effect, as WT mice receiving two GnRH pulses displayed LH responses approximately 2-fold greater than those receiving only one pulse. By contrast, PRKO mice receiving two GnRH pulses exhibited no additional increase in plasma LH levels. We conclude that PR activation is obligatory for expression of the GnRH self-priming effect as well as for generation of E2-induced LH and FSH surges. The extent to which failure of LH surge secretion in PRKO mice is due to the absence of GnRH self-priming, lack of hypothalamic GnRH surges, and/or defects in other processes remains to be determined. These observations clearly demonstrate, however, that the presence of PR is an absolute requirement for the transmission of E2-induced signals leading to gonadotropin surges.