Control Of GnRH Secretion Research Articles

Kisspeptin and neurokinin B neuroendocrine pathways in the control of human ovulation.

The roles of initially kisspeptin and subsequently neurokinin B pathways in the regulation of human reproduction through the control of GnRH secretion were first identified 20 years ago, as essential for the onset of puberty in both boys and girls. Within that short time we already now have the first licence for clinical use for a neurokinin antagonist in a related indication, for menopausal vasomotor symptoms. Between these two markers of the start and end of the reproductive lifespan, it is clear that these pathways underlie many of the aspects of the hypothalamic regulation of reproduction which had hitherto been enigmatic. In this review, we describe the data currently available from studies designed to elucidate the roles of kisspeptin and neurokinin B in human ovarian function, specifically the regulation of follicle development leading up to ovulation, and in the control of the mid-cycle GnRH/LH surge that triggers ovulation. These studies, undertaken with only very limited pharmacological tools, provide evidence that the neurokinin B pathway is important in controlling the hypothalamic contribution to the precise gonadotropic drive to the ovary that is necessary for mono-ovulation, whereas the switch from negative to positive estrogenic feedback results in kisspeptin-mediated increased GnRH secretion. Potential therapeutic opportunities in conditions characterised by disordered hypothalamic/pituitary function, polycystic ovary syndrome, and functional hypothalamic amenorrhoea, and in the induced LH surge that is a necessary part of IVF treatment are discussed.

LBMON114 Enrichment Of Rare Sequence Variants In Genes That Communicate Metabolic Signals To The GnRH System In Hypothalamic Amenorrhea

Abstract Introduction Functional hypothalamic amenorrhea (HA) is commonly associated with increased exercise or decreased caloric intake and often with stress. We have previously demonstrated an increased burden of rare sequence variants (RSVs) in genes involved in GnRH ontogeny and upstream regulation in women with HA, but the role of metabolic and stress signaling to the GnRH neuronal system is poorly defined in this population. Methods The study included 100 women with a confirmed diagnosis of HA. The control cohort consisted of 468 women (aged 45­­-65 years) drawn from the NIH ClinSeq® Project. Exome sequencing was performed on peripheral blood genomic DNA. A subset of 72 genes was analyzed that have been shown to: 1) link metabolic or stress with reproductive phenotypes or 2) integrate metabolic and stress pathways with control of GnRH secretion. Joint genotyping of case and control samples was performed using the GATK GenotypeGVCFs function, locus-filtering using the VariantRecalibrator function, and genotype refinement using CalculateGenotypePosteriors with computation of median depths. Median depth positions <10, positions failing GATK VQSR or GATK genotype quality scores <20 were excluded. RSVs were identified by < 1% frequency in any subpopulation in gnomeAD for all-subjects (AS) and < 1% frequency in non-Finnish Europeans for Caucasians (CS). Data were analyzed for AS and for CS using a one-sided Fisher exact test for metabolism genes and stress genes. An additional regression analysis was conducted on the number of RSVs in a given gene as a predictor of HA vs. control. Comparisons with a p-value of < 0.1 are reported. Results HA patients exhibited an increased burden of RSVs in metabolism genes vs. controls (AS p=0. 043; CS p=0.105). The total number of RSVs per gene highlighted differences between HA and controls for the following genes: ADAMTSL1, GRINA, GRIN1, HCRTR1, TENM3, and NOS1 (AS p<0. 001, p=0. 032, p=0. 057, p=0. 082, p=0. 091, p=0. 095; CS p=0. 024, p=0. 044, p=0. 044, p NS, p=0. 086, p NS). Interestingly, RSVs in NOS1 and TENM3 appeared to be protective for HA (odds ratio <1 for both). In contrast, candidate stress genes were not significant in either the AS or CS (p=0.788, p=0.910). Conclusions These data suggest that RSVs in genes involved in phenotypes or signaling pathways that link metabolism to GnRH secretion may predispose to development of HA in the setting of decreased energy balance, but not for stress-related genes. GRINA and GRIN1 are important components of glutamate signaling that facilitate both appetite and GnRH secretion either directly or through kisspeptin. HCRTR1 plays a similar role in linking appetite and GnRH secretion while NOS1, which facilitates kisspeptin signaling, may be protective. This work highlights the need for further studies to understand the potential roles of ADAMTSL1 and TENM3 as risk factors for HA. Presentation: Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

Control of GnRH secretion

Control of GnRH secretion

Unraveling the connection between GABA and kisspeptin in the control of reproduction.

Neuroendocrine control of reproduction involves the interplay of various factors that become active at some point along development. GnRH is the main neurohormone controlling reproduction and among the most important inputs modulating GnRH synthesis/secretion are GABA and kisspeptins. These interactions of GABA and kisspeptin in the control of GnRH secretion can take place by the presence of the receptors of both factors on the GnRH neuron or alternatively by the actions of GABA on kisspeptin neurons and/or the actions of kisspeptin on GABA neurons. Kisspeptin acts on the Kiss1R, a seven transmembrane domain, Gαq/11-coupled receptor that activates phospholipase C, although some Gαq/11-independent pathways in mediating part of the effects of Kiss1R activation have also been proposed. GABA acts through two kinds of receptors, ionotropic GABAA/C receptors involving a chloride channel and associated with fast inhibitory/stimulatory conductance and metabotropic GABAB receptors (GABABR) that are Gi/0 protein linked inducing late slow hyperpolarization. In this review, we aim to summarize the different ways in which these two actors, kisspeptin and GABA, interact to modulate GnRH secretion across the reproductive lifespan.

Gamma amino-butyric acid and the control of GnRH secretion in sheep

The release of GnRH from nerve terminals in the median eminence into the portal vessels is influenced by factors in the internal and external environment of the animal. In the former category are the gonadal steroid hormones oestrogen and progesterone which alter the characteristics of GnRH secretion during the oestrous and seasonal cycles. These cannot exert their actions directly on the GnRH neurones as they do not possess hormone receptors. Therefore, some other steroid-sensitive neuronal system must relay this information to the GnRH neurones. Gamma amino-butyric acid (GABA) neurones are good candidates for this role as they contain steroid hormone receptors and synapse on GnRH neurones. Recent studies in ewes have sought to identify a role for GABA in mediating the actions of both oestrogen and progesterone on GnRH release. The technique of microdialysis was used to monitor GABA concentrations in areas containing GnRH cell bodies during the oestrogen-induced surge of GnRH and during progesterone negative feedback. Concentrations of this inhibitory neurotransmitter have been shown to fall in the former situation where GnRH release is being stimulated, but to be increased when progesterone is depressing GnRH release. GABA may also be important in mediating the seasonal switch in the negative feedback actions of oestradiol. During the anoestrous season, when oestradiol is a potent inhibitor of GnRH secretion, specific GABA receptor antagonists can stimulate neurohormone release, an action that is not observed in the breeding season when oestrogen is much less potent.

Kisspeptin neuronal networks in pubertal development of domestic female ruminants

Summary. The pubertal activation of high-frequency episodic pulses of GnRH is believed to occur as a result of a change in the balance between inhibitory and excitatory stimuli to GnRH neurons. Kisspeptin neurons have been identified as major components of the pathway that regulates GnRH neuronal activity and appear to mediate the effects of estradiol in the control of GnRH secretion. The influence of nutrition on timing the onset of puberty may also involve kisspeptin neurons. Neuropeptide Y (NPY) and proopiomelanocortin (POMC) neurons in the hypothalamus interact with kisspeptin neurons in a network that is likely to mediate the nutritional and gonadal steroid regulation of reproductive function. Pre- and postnatal programming of these neuronal networks may be a primary mechanism by which nutrition and endocrine factors time the onset of a pubertal pattern of GnRH secretion. The genetic components underlying the function of these networks have begun to be revealed with the availability of high- throughput technologies and computational tools. A complex, highly interactive network of regulatory genes appears upstream to KISS1 and probably involves multiple cellular phenotypes. Characterization of the cellular location, temporal activation, and biological function of the various molecular components of the genetic network that regulate kisspeptin neuronal pathways will be essential for a full understanding of the role of KISS1 in the process of pubertal maturation.

Melatonin binding sites and their role in seasonal reproduction

The pineal gland has a major role in the translation of scotophase duration into a hormonal signal by the hormone melatonin. Animals such as sheep, goats and deer use the seasonal variation of this signal to coordinate reproductive behaviour with the environment. Despite intensive research over more than 30 years the site(s) of action of melatonin and the resultant intracellular responses are still not clear. This review discusses recent work that has localized the site of action of melatonin in sheep using administration into the hypothalamus in vivo as well as studies on putative melatonin receptors in the pars tuberalis and brain. There is clear evidence that melatonin acting at the level of the pars tuberalis is involved in the seasonal regulation of prolactin secretion, but the evidence for involvement of the pars tuberalis in seasonal reproduction is not compelling. Localized administration of melatonin to the sheep brain revealed that areas anatomically distinct from the pars tuberalis, the ventromedial and arcuate nuclei, simulated seasonal reproductive changes in rams and ewes. Recent studies on brain melatonin binding sites in our laboratory have shown that an antagonist of tissue transglutaminase, Bacitracin, as well as substrates for the enzyme inhibit binding of melatonin to brain membranes. As a working hypothesis, we propose that pineal melatonin secretion alters seasonal reproduction by interactions with a neural transglutaminase at the synapse of neurones involved in the control of GnRH secretion. Synaptic transglutaminase is implicated in the control of the release of neurotransmitter via the synaptic vesicle associated protein, synapsin 1; activation of transglutaminase results in the covalent modification of synapsin 1 such that vesicles are not released from the cytoskeleton. Seasonal variation in the duration of melatonin secretion may result in similar variations in the duration of suppression and activation of transglutaminase. The resultant changes in transmitter release may then be responsible for the seasonal neuronal plasticity previously observed in GnRH neurones.

The versatile tanycyte: a hypothalamic integrator of reproduction and energy metabolism

This review is interesting for several reasons. It focuses on a specific type of cells lining the wall of the third ventricle in the median eminence of the hypothalamus. This region acts both as a secretor of neuropeptides and as a sensor of the metabolic status of the periphery. These two functions imply a bidirectional physiological pathway: brain-to-periphery and vice-versa; different cellular mechanisms have been described. The interest developed for the last few years on the function of tanycytes in normal brain and their possible dysfunctions in neuroendocrine disorders has been justified by the importance to increase our knowledge on the control of the homeostasis by the brain. It is now obvious that tanycytes are involved in the control of key hypothalamic functions, such as reproduction and energy metabolism. These authors review recent evidence on the different types of tanycytes in the hypothalamus, their role in the blood-brain barrier, and also in the control of GnRH secretion. One paragraph is dedicated to the role of tanycytes in hypothalamic neurogenesis. This recent discovery was completely unexpected and opens a new area in neuroendocrine research. In the last part of this review, the authors discuss that tanycytes may be an interesting new therapeutic target not only in reproduction, but also in obesity and aging.

Reproductive parameters of female Wistar rats treated with methylphenidate during development

Methylphenidate (MPH), a psychoactive agent that acts mainly by blocking the uptake of dopamine, is the main drug used to treat Attention Deficit Hyperactivity Disorder in children and adolescents. During development, important changes in brain architecture and plasticity occur, these changes, sensitive to exposure to stimulant drugs, are important in the control of GnRH secretion, influencing the release of sex hormones throughout the ovarian cycle. This study investigated the effects of repeated treatment with MPH during development on reproductive parameters of adult female rats. Wistar rats received MPH 2.5mg/kg, MPH 5.0mg/kg, or tap water (gavage) from postnatal day (PND) 21 to PND 60. From PND 75, one subgroup of females was selected for evaluation of estrous cycle, estradiol levels, weight of sexual organs, and histomorphological analysis of ovary follicles and uterus. In another subgroup, the sexual and maternal behaviors were evaluated at PND 90 and on lactational day 5, respectively. No significant alterations were observed in the MPH groups. This study demonstrated that repeated administration of MPH during the period corresponding to childhood to early adulthood does not interfere in the reproductive function of female rats in adulthood.

Curiouser and Curiouser: The Evolving Story of the Mechanisms Involved in Puberty.

Sometimes, like a giant jigsaw puzzle, concepts in endocrinology take time to develop, to form a coherent picture of how endocrine systems are integrated to support physiological responses. Such, it can be argued, has been the case with respect to our understanding of the mechanisms underlying puberty. For more than 50 years, it has been known that mammals experience a period of agonadal restraint during development, in which reproductive function is held in check (1). As puberty approaches, this restraint is switched off and pulsatile GnRH secretion is activated. What caused the juvenile period to end remained a matter of conjecture, although a shift in steroid feedback was suggested by observations showing that immature rats had enhanced sensitivity to the negative feedback effects of estradiol (2), whereas the onset of ovarian cyclicity could be advanced by prepubertal injections of estradiol (3). In the past 5 decades, several other important pieces of the puzzle have been filled in. The brain was itself found to be a site of gonadal steroid synthesis (4, 5). Data emerged to indicate that neurosteroid synthesis might contribute to reproductive function (6). Dramatic advances were made in understanding the neurotransmitter and neuropeptide circuitry responsible for regulating the onset of reproductive function, in particular the kisspeptin system (7, 8). However, what actually triggered the change in feedback control of gonadotrophin secretion at puberty remained obscure. A shift from a predominantly inhibitoryhypothalamic environment toone inwhichexcitatory neurotransmitters activated the GnRH neurons was believed to occur (9, 10), but what caused this shift remained unknown. In the current issue of Endocrinology, Kenealy et al (11) develop the novel idea that in female primates, the control of GnRH secretion before and during puberty may actually operate in a relatively similar hypothalamic steroidal milieu: only the source of the estradiol is different. To determine hypothalamic steroid concentrations, perfusates were collected in vivo from the stalk-median eminence (S-ME) region of 32 female rhesus monkeys at different stages of prepubertal and pubertal development. The samples were assayed for estradiol, estrone, testosterone, androstenedione, and progesterone, using a sensitive and specific liquid chromatography-mass spectrometry-based method. Concomitantly, GnRH in the perfusates, as well as LH and steroid levels in circulating serum samples from the same animals, were also assayed. As expected, GnRH and LH levels followed a similar developmental pattern, levels at midpuberty being higher than either prepuberty or early puberty. Circulating estradiol levels also were higher in midpuberty. The steroid measurements in the S-ME perfusates, however, exhibited a strikingly different pattern. Although androstenedione, testosterone, and progesterone did not change significantly in the perfusates across the different stages of puberty, estrone and estradiol were found to be high in the S-ME perfusates from prepubertal animals, declining in midpuberty and early puberty, respectively. Because serum estradiol levels increased over the same period, the S-ME to serum ratio fell dramatically in the midpubertal animals, more than 5-fold in the case of estradiol and more than 60-fold in the case of estrone. These results suggest that although the prepubertal period is indeed characterized by a period of GnRH inhibition that does not depend on ovarian hormone secretion, it is not in fact steroid independent. Rather, it may be mediated at least in part by estradiol produced in situ,

Activation of Neurokinin 3 Receptors Stimulates GnRH Release in a Location-Dependent but Kisspeptin-Independent Manner in Adult Mice

GnRH neurons form the final common pathway for the central control of reproduction. GnRH release occurs from terminals in the external layer of the median eminence (ME) for neuroendocrine control of the pituitary, and near GnRH-GnRH fiber appositions within the preoptic area (POA). Whether or not control of GnRH secretion by neuromodulators is different in these 2 areas is unknown. Mutations in neurokinin B (NKB) or the neurokinin-3 receptor (NK3R) are linked to hypogonadotropic hypogonadism in humans, suggesting that NKB may regulate GnRH secretion. Using fast scan cyclic voltammetry through carbon-fiber microelectrodes, we examined real-time GnRH release in response to the NK3R agonist senktide in the ME and POA. Coronal brain slices were acutely prepared from adult gonad-intact GnRH-green fluorescent protein male mice, and carbon-fiber microelectrodes were placed either within green fluorescent protein-positive terminal fields of the ME or near GnRH-GnRH fiber appositions in the POA. Senktide induced GnRH release consistently in the ME but not the POA, indicating that GnRH release is differentially regulated by NKB in a location-dependent manner. Senktide also induced GnRH secretion in the ME of kisspeptin-knockout (Kiss1 knockout) mice. Interestingly, release amplitude was lower compared with wild-type mice. These data indicate regulation of GnRH release by NK3R agonists is site specific and suggest that kisspeptin is not a required mediator between NK3R activation and GnRH secretion in the ME. This information will be useful for informing future models of afferent regulation of GnRH release.

The effect of chronic kisspeptin administration on seminal fructose levels in male mice

The discovery that kisspeptin was critical for normal fertility in all mammalian species including humans, ushered in a new chapter in our understanding of the control of GnRH secretion. Kisspeptin, the product of the KISS1 gene, plays an essential role in the regulation of spermatogenesis acting primarily at the hypothalamic level of the gonadotropic axis. Among the many identified substances in human semen, fructose is becoming increasingly significant. Fructose is synthesized and secreted by the seminal vesicles. Its synthesis is regulated by androgens and it is correlated directly with the levels of testosterone. Dose dependent degeneration of seminal vesicle has been described following intraperitoneal kisspeptin treatment; however, effects of kisspeptin administration on the levels of seminal fructose remain elusive till date. The present study, therefore, addresses the effects of 12-day administration of kisspeptin on seminal fructose levels in male mice. Kisspeptin-10 was administered intraperitoneally at different dosage concentrations (1 μg, 1 ng, and 10 ρg) to adult male mice, twice daily for 12 days. Seminal fructose levels were studied photometrically after 12 days of treatment. At the end of the treatment, seminal fructose levels decreased significantly after all tested doses. Chronic intermittent kisspeptin-10 administration negatively regulates seminal fructose levels in adult male mice.

Neuropeptide Y induces gonadotropin-releasing hormone gene expression directly and through conditioned medium from mHypoE-38 NPY neurons

Neuropeptide Y (NPY) regulates reproductive function at the level of the hypothalamus through control of GnRH secretion. However, the direct control of GnRH gene expression by NPY has not yet been studied. GT1-7 neurons were treated with 100 nM of NPY over a 36 h time course. GnRH mRNA levels were significantly increased by NPY up to 12 h. We determined that GT1-7 neurons expressed Y1, Y2, and Y4 NPY receptors, but not Y5. Functional analysis of NPY receptor activation indicated that the Y1/Y4/Y5 receptor agonist [Leu31, Pro34] significantly induced cAMP accumulation in the GT1-7 neurons. Western blot studies demonstrated changes in the phosphorylation status of AKT, ERK1/2, CREB and ATF-1 after NPY exposure. Pharmacological inhibitors of the MAPK and PKA signal transduction pathways attenuated the NPY-mediated increase in GnRH transcription. This NPY-mediated increase in GnRH mRNA was also inhibited with the Y1-receptor specific antagonist BIBP-3226. The mHypoE-38 neurons secrete detectable levels of NPY and can be used as an endogenous source of NPY. Conditioned medium from mHypoE-38 neurons induced an increase in GnRH mRNA, which was inhibited by the Y1 receptor antagonist BIBP-3226. Together, these studies strengthen the evidence for the importance of NPY in the regulation of reproductive function.

Basic Aspects of the Control of GnRH and LH Secretions by Kisspeptin: Potential Applications for Better Control of Fertility in Females

The neuronal control of fertility and sterility has been a subject of research for years. However, nowadays, in spite of considerable literature about GnRH during the last few decades, the precise cellular and molecular mechanisms whereby gonadal steroids and other peripheral signals converge in the brain to achieve the fine regulation of GnRH secretion remains partially unknown. In this scenario, a major breakthrough in our understanding of the neuronal signals governing reproduction took place in 2003 with the discovery of metastin/kisspeptin as a major player in the control of GnRH secretion. This molecule, first described as having a crucial role in triggering the onset of puberty, is involved in all phases of reproductive life and hence has attracted the interest of many reproductive neuroendocrinologists. Administered either centrally or peripherally, kisspeptin strongly induces the secretion of gonadotropin in many species, mainly through stimulation of GnRH secretion. Kisspeptin cells involved in the control of GnRH secretion are located in two regions of the brain: the preoptic area and the arcuate nucleus. Carrying oestradiol receptor alpha, kisspeptin cells of these regions appear to be the main integration centres for the expression of both the positive and negative feedback of steroid on GnRH secretion. More recently, this molecule has been shown to be able to synchronize preovulatory surges in cyclic ewes and cause ovulation in seasonally acyclic ewes. This review summarizes the most relevant aspects of the role of kisspeptin in GnRH/LH release and the potential application of this molecule in new strategies for controlling female fertility.

Sexual Differentiation of the External Genitalia and the Timing of Puberty in the Presence of an Antiandrogen in Sheep

Testicular steroids during midgestation sexually differentiate the steroid feedback mechanisms controlling GnRH secretion in sheep. To date, the actions of the estrogenic metabolites in programming neuroendocrine function have been difficult to study because exogenous estrogens disrupt maternal uterine function. We developed an approach to study the prenatal actions of estrogens by coadministering testosterone (T) and the androgen receptor antagonist flutamide, and tested the hypothesis that prenatal androgens program estradiol inhibitory feedback control of GnRH secretion to defeminize (advance) the timing of the pubertal increase in LH. Pregnant sheep were either untreated or treated with T, dihydrotestosterone (DHT) (a nonaromatizable androgen), or T plus flutamide from d 30-90 of gestation. To study the postnatal response to steroid negative feedback, lambs were gonadectomized and estradiol-replaced, and concentrations of LH were monitored in twice-weekly blood samples. Although T and DHT produced penile and scrotal development in females, the external genitalia of T plus flutamide offspring remained phenotypically female, regardless of genetic sex. Untreated females and females and males treated with T plus flutamide exhibited a pubertal increase in circulating LH at 26.4+/-0.5, 26.0+/-0.7, and 22.4+/-1.6 wk of age, respectively. In females exposed to prenatal androgens, the LH increase was advanced (T: 12.0+/-2.6 wk; DHT: 15.0+/-2.6 wk). These results demonstrate the usefulness of combining T and antiandrogen treatments as an approach to increasing prenatal exposure to estradiol. Importantly, the findings support our hypothesis that prenatal androgens program sensitivity to the negative feedback actions of estradiol and the timing of neuroendocrine puberty.

Activation of Neuronal Nitric Oxide Release Inhibits Spontaneous Firing in Adult Gonadotropin-Releasing Hormone Neurons: A Possible Local Synchronizing Signal

The activation of nitric oxide (NO) signaling pathways in hypothalamic neurons plays a key role in the control of GnRH secretion that is central to reproductive function. It is unknown whether NO directly modulates the firing behavior of GnRH neurons in the preoptic region of the mature brain. Using patch-clamp recordings from GnRH neurons expressing green fluorescent protein in adult mice brain slices, we demonstrate that the NO precursor, L-arginine (Arg), or the NO donor, diethylamine/NO, induced a robust and reversible reduction in the spontaneous firing activity of GnRH neurons, including bursting activity. The effects of L-Arg were prevented by the NO synthase inhibitor N omega-nitro-L-Arg methyl ester hydrochloride. Histochemical studies revealing a close anatomical relationship between neurons producing NO and GnRH perikarya, together with the loss of the L-Arg-mediated inhibition of GnRH neuronal activity via the selective blockade of neuronal NO synthase, suggested that the primary source of local NO production in the mouse preoptic region was neuronal. Synaptic transmission uncoupling did not alter the effect of NO, suggesting that NO affects the firing pattern of GnRH neurons by acting at a postsynaptic site. We also show that the NO-mediated changes in membrane properties in the GnRH neurons require soluble guanylyl cyclase activity and may involve potassium conductance. By revealing that NO is a direct modulator of GnRH neuronal activity, our results introduce the intriguing possibility that this gaseous neurotransmitter may be used by the sexual brain to modulate burst firing patterns. It may set into phase the bursting activity of GnRH neurons at key stages of reproductive physiology.

Orphanin FQ: Evidence for a Role in the Control of the Reproductive Neuroendocrine System

Orphanin FQ (OFQ), also known as nociceptin, is a member of the endogenous opioid peptide family that has been functionally implicated in the control of pain, anxiety, circadian rhythms, and neuroendocrine function. In the reproductive system, endogenous opioid peptides are involved in the steroid feedback control of GnRH pulses and the induction of the GnRH surge. The distribution of OFQ in the preoptic area and hypothalamus overlaps with GnRH, and in vitro evidence suggests that OFQ can inhibit GnRH secretion from hypothalamic fragments. Using the sheep as a model, we examined the potential anatomical colocalization between OFQ and GnRH using dual-label immunocytochemistry. Confocal microscopy revealed that approximately 93% of GnRH neurons, evenly distributed across brain regions, were also immunoreactive for OFQ. In addition, almost all GnRH fibers and terminals in the external zone of the median eminence, the site of neurosecretory release of GnRH, also colocalized OFQ. This high degree of colocalization suggested that OFQ might be functionally important in controlling reproductive endocrine events. We tested this possibility by examining the effects of intracerebroventricular administration of [Arg(14), Lys(15)] OFQ, an agonist to the OFQ receptor, on pulsatile LH secretion. The agonist inhibited LH pulse frequency in both luteal phase and ovariectomized ewes and suppressed pulse amplitude in the latter. The results provide in vivo evidence supporting a role for OFQ in the control of GnRH secretion and raise the possibility that it acts as part of an ultrashort, autocrine feedback loop controlling GnRH pulses.

Novel concepts about normal sexual differentiation of reproductive neuroendocrine function and the developmental origins of female reproductive dysfunction: the sheep model

The neuroendocrine regulation of GnRH secretion plays a central role in timing gamete release in both sexes. This regulation is more complex in the female because the discontinuous release of ova is more complex than the continuous release of spermatozoa. This review provides an evolving understanding of the sex differences in reproductive neuroendocrine controls and how these differences arise. The rules for sexual differentiation of steroid feedback control of GnRH secretion conceptually parallel the well-established principles that underlie the sexual differentiation of the internal and external genitalia. In the context of the neuroendocrine regulation of the ovarian cycle, and using the sheep as a model, four steroid feedback controls for GnRH secretion are inherent (default). They require no ovarian developmental input to function appropriately during adulthood. Two steroid feedback controls regulate the preovulatory surge mode of GnRH secretion, and two regulate the pulsatile mode. If the individual is a male, three steroid feedback controls of GnRH secretion become unnecessary or irrelevant, and these are abolished or become functionally inoperative through programmed reductions in hypothalamic sensitivity. This central programming occurs through exposure of presynaptic GnRH neurons in the developing male brain to the androgenic and estrogenic actions of testicular steroids. In precocial species such as ruminants, this programming begins well before birth. Understanding how GnRH secretion normally becomes sexually differentiated is of practical importance to determining how inappropriate hormonal environments during development can variously malprogram the neuroendocrine system to produce a variety of reproductive dysfunctions relating to patterning of gonadotropin secretion.

Expression of feeding-related peptide receptors mRNA in GT1-7 cell line and roles of leptin and orexins in control of GnRH secretion1

To investigate the expression of feeding-related peptide receptors mRNA in GT1-7 cell line and roles of leptin and orexins in the control of GnRH secretion. Receptors of bombesin3, cholecystokinin (CCK)-A, CCK-B, glucagon-like peptide (GLP)1, melanin-concentrating hormone (MCH)1, orexin1, orexin2, neuromedin-B, neuropeptide Y (NPY)1 and NPY5, neurotensin (NT)1, NT2, NT3, and leptin receptor long form mRNA in GT1-7 cells were detected by reversed transcriptase-polymerase chain reaction. GT1-7 cells were treated with leptin, orexin A and orexin B at a cohort of concentrations for different lengths of time, and GnRH in medium was determined by radioimmunoassay (RIA). Receptors of bombesin 3, CCK-B, GLP1, MCH1, orexin1, neuromedin-B, NPY1, NPY5, NT1, NT3, and leptin receptor long form mRNA were expressed in GT1-7 cells, of which, receptors of GLP1, neuromedin-B, NPY1, and NT3 were highly expressed. No amplified fragments of orexin2, NT2, and CCK-A receptor cDNA were generated with GT1-7 RNA, indicating that the GT1-7 cells did not express mRNA of them. Leptin induced a significant stimulation of GnRH release, the results being most significant at 0.1 nmol/L for 15 min. In contrast to other studies in hypothalamic explants, neither orexin A nor orexin B affected basal GnRH secretion over a wide range of concentrations ranging from 1 nmol/L to 500 nmol/Lat 15, 30, and 60 min. Feeding and reproductive function are closely linked. Many orexigenic and anorexigenic signals may control feeding behavior as well as alter GnRH secretion through their receptors on GnRH neurons.

Influence of estradiol on NADPH diaphorase/neuronal nitric oxide synthase activity and colocalization with progesterone or type II glucocorticoid receptors in ovine hypothalamus.

Nitric oxide (NO) has been shown to play an important role in both the neuroendocrine reproductive and stress axes, which are closely linked. Because progesterone (P4) receptors (PRs) and glucocorticoid receptors (GRs) are not found in GnRH neurons and the NOergic system has been implicated in the control of GnRH secretion, this study aimed to ascertain whether steroids altered the NOergic system. Our first objective was to map the distribution of NO synthase (NOS) cells in the ovine preoptic area (POA) and hypothalamus and to determine whether NOS activity is enhanced by estradiol (E2) treatment. Using NADPH diaphorase (NADPHd) histochemistry, we found that NADPHd-positive neurons were spread throughout the ovine POA and hypothalamus, and that all NADPHd cells were immunoreactive for NOS. In response to estradiol, a significant increase in the number of NADPHd cells was noted only in the ventrolateral region of the ventromedial nucleus (VMNvl), with no significant difference in the POA or arcuate nucleus. Progesterone and glucocorticoid receptors were colocalized with NADPHd reactive neurons in the POA, arcuate nucleus, and VMNvl of ewes in both treatment groups. In ewes receiving estradiol, the number of NADPHd-positive cells containing steroid receptors in the POA (PR, 81%; GR, 79%) and arcuate nucleus (PR, 89%; GR, 84%) was similar, but in the VMNvl, fewer NADPHd-positive cells contained GR (PR, 88%, GR, 31%). These data show that estradiol up-regulates NOS activity in a site-specific manner and that the influence and possible interaction of progesterone and corticosteroids on NO producing cells may differ according to the neural location.