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Puberty is the process of achieving the capacity for reproduction and depends upon increased gonadotropin-releasing hormone (GnRH) secretion. Neurons in the arcuate nucleus of the hypothalamus (ARC) coexpressing kisspeptin, neurokinin B (NKB), and dynorphin (i.e. KNDy cells) are critical for puberty onset. In humans and livestock, mistimed puberty has negative consequences for health and production, respectively. Puberty is largely impacted by genetics and genes associated with its timing include those for kisspeptin and NKB. Epigenetic modification of genes and transcriptional activity also influence puberty onset. Adequate nutrition is required for normal puberty onset and the hormone leptin links metabolic status and puberty. Leptin acts through ARC neurons reciprocally connected with KNDy neurons that transduce nutritional signals into inputs influencing GnRH neurons. Further work using multiple animal models, including agriculturally important species, will be necessary for understanding the neural mechanisms underlying puberty onset and the problems associated with mistimed puberty.

Neurones in the arcuate nucleus co-expressing kisspeptin, neurokinin B (NKB) and dynorphin (KNDy) play a critical role in the control of gonadotrophin-releasing hormone (GnRH) and luteinising hormone (LH) secretion. In sheep, KNDy neurones mediate both steroid-negative- and -positive-feedback during pulsatile and preovulatory surge secretions of GnRH/LH, respectively. In addition, KNDy neurones receive glutamatergic inputs expressing vGlut2, a glutamate transporter that serves as a marker for those terminals, from both KNDy neurones and other populations of glutamatergic neurones. Previous work reported higher numbers of vGlut2-positive axonal inputs onto KNDy neurones during the LH surge than in luteal phase ewes. In the present study, we further examined the effects of the ovarian steroids progesterone (P) and oestradiol (E2 ) on glutamatergic inputs to KNDy neurones. Ovariectomised (OVX) ewes received either no further treatment (OVX) or steroid treatments that mimicked the luteal phase (low E2 + P), and early (low E2 ) or late follicular (high E2 ) phases of the oestrous cycle (n = 4 or 5 per group). Brain sections were processed for triple-label immunofluorescent detection of NKB/vGlut2/synaptophysin and analysed using confocal microscopy. We found higher numbers of vGlut2 inputs onto KNDy neurones in high E2 compared to the other three treatment groups. These results suggest that synaptic plasticity of glutamatergic inputs onto KNDy neurones during the ovine follicular phase depend on increasing levels of E2 required for the preovulatory GnRH/surge. These synaptic changes likely contribute to the positive-feedback action of oestrogen on GnRH/LH secretion and thus the generation of the preovulatory surge in the sheep.

Neurokinin B (NKB) is critical for fertility in humans and stimulates gonadotrophin-releasing hormone/luteinising hormone (LH) secretion in several species, including sheep. There is increasing evidence that the actions of NKB in the retrochiasmatic area (RCh) contribute to the induction of the preovulatory LH surge in sheep. In the present study, we determined whether there are sex differences in the response to RCh administration of senktide, an agonist to the NKB receptor (neurokinin receptor-3 [NK3R]), and in NKB and NK3R expression in the RCh of sheep. To normalise endogenous hormone concentrations, animals were gonadectomised and given implants to mimic the pattern of ovarian steroids seen in the oestrous cycle. In females, senktide microimplants in the RCh produced an increase in LH concentrations that lasted for at least 8 hours after the start of treatment, whereas a much shorter increment (approximately 2 hours) was seen in males. We next collected tissue from gonadectomised lambs 18 hours after the insertion of oestradiol implants that produce an LH surge in female, but not male, sheep for immunohistochemical analysis of NKB and NK3R expression. As expected, there were more NKB-containing neurones in the arcuate nucleus of females than males. Interestingly, there was a similar sexual dimorphism in NK3R-containing neurones in the RCh, NKB-containing close contacts onto these RCh NK3R neurones, and overall NKB-positive fibres in this region. These data demonstrate that there are both functional and morphological sex differences in NKB-NK3R signalling in the RCh and raise the possibility that this dimorphism contributes to the sex-dependent ability of oestradiol to induce an LH surge in female sheep.

Elevated and sustained estradiol concentrations cause a gonadotropin-releasing hormone (GnRH) and luteinizing hormone (LH) surge that is necessary for ovulation. In sheep, several different neural systems have been implicated in this stimulatory action of estradiol and this study focused on somatostatin (SST) neurons in the ventral lateral region of the ventral medial nucleus (vlVMN) which express c-Fos during the surge. First, we determined if increased activity of SST neurons could be related to elevated GnRH secretion by assessing SST synapses onto GnRH neurons and neurons coexpressing kisspeptin, neurokinin B, dynorphin (KNDy). We found that the percentage of preoptic area GnRH neurons that receive SST input increased during the surge compared with other phases of the cycle. However, since SST is generally inhibitory, and pharmacological manipulation of SST signaling did not alter the LH surge in sheep, we hypothesized that nitric oxide (NO) was also produced by these neurons to account for their activation during the surge. In support of this hypothesis we found that (1) the majority of SST cells in the vlVMN (>80%) contained neuronal nitric oxide synthase (nNOS); (2) the expression of c-Fos in dual-labeled SST-nNOS cells, but not in single-labeled cells, increased during the surge compared with other phases of the cycle; and (3) intracerebroventricular (ICV) infusion of the nitric oxide synthase inhibitor, N(G)-nitro-L-arginine methyl ester, completely blocked the estrogen-induced LH surge. These data support the hypothesis that the population of SST-nNOS cells in the vlVMN are a source of NO that is critical for the LH surge, and we propose that they are an important site of estradiol positive feedback in sheep.

Early work in ewes provided a wealth of information on the physiological regulation of pulsatile gonadotropin-releasing hormone (GnRH) secretion by internal and external inputs. Identification of the neural systems involved, however, was limited by the lack of information on neural mechanisms underlying generation of GnRH pulses. Over the last decade, considerable evidence supported the hypothesis that a group of neurons in the arcuate nucleus that contain kisspeptin, neurokinin B and dynorphin (KNDy neurons) are responsible for synchronizing secretion of GnRH during each pulse in ewes. In this review, we describe our current understanding of the neural systems mediating the actions of ovarian steroids and three external inputs on GnRH pulsatility in light of the hypothesis that KNDy neurons play a key role in GnRH pulse generation. In breeding season adults, estradiol (E2) and progesterone decrease GnRH pulse amplitude and frequency, respectively, by actions on KNDy neurons, with E2decreasing kisspeptin and progesterone increasing dynorphin release onto GnRH neurons. In pre-pubertal lambs, E2inhibits GnRH pulse frequency by decreasing kisspeptin and increasing dynorphin release, actions that wane as the lamb matures to allow increased pulsatile GnRH secretion at puberty. Less is known about mediators of undernutrition and stress, although some evidence implicates kisspeptin and dynorphin, respectively, in the inhibition of GnRH pulse frequency by these factors. During the anoestrus, inhibitory photoperiod acting via melatonin activates A15 dopaminergic neurons that innervate KNDy neurons; E2increases dopamine release from these neurons to inhibit KNDy neurons and suppress the frequency of kisspeptin and GnRH release.

A subpopulation of neurons located within the arcuate nucleus, colocalizing kisspeptin, neurokinin B, and dynorphin (Dyn; termed KNDy neurons), represents key mediators of pulsatile GnRH secretion. The KNDy model of GnRH pulse generation proposes that Dyn terminates each pulse. However, it is unknown where and when during a pulse that Dyn is released to inhibit GnRH secretion. Dyn acts via the κ opioid receptor (KOR), and KOR is present in KNDy and GnRH neurons in sheep. KOR, similar to other G protein–coupled receptors, are internalized after exposure to ligand, and thus internalization can be used as a marker of endogenous Dyn release. Thus, we hypothesized that KOR will be internalized at pulse termination in both KNDy and GnRH neurons. To test this hypothesis, GnRH pulses were induced in gonad-intact anestrous ewes by injection of neurokinin B (NKB) into the third ventricle and animals were euthanized at times of either pulse onset or termination. NKB injections produced increased internalization of KOR within KNDy neurons during both pulse onset and termination. In contrast, KOR internalization into GnRH neurons was seen only during pulse termination, and only in GnRH neurons within the mediobasal hypothalamus (MBH). Overall, our results indicate that Dyn is released onto KNDy cells at the time of pulse onset, and continues to be released during the duration of the pulse. In contrast, Dyn is released onto MBH GnRH neurons only at pulse termination and thus actions of Dyn upon KNDy and GnRH cell bodies may be critical for pulse termination.

Seasonal reproduction in sheep is primarily due to a dramatic increase in the ability of estradiol (E2) to inhibit the pulsatile secretion of gonadotropin-releasing hormone (GnRH) during the nonbreeding season [anestrus (ANS)]. Recent findings suggest that kisspeptin/neurokinin B/dynorphin (KNDy) neurons of the arcuate nucleus (ARC) play a key role in conveying this negative feedback influence, with dopaminergic projections from the retrochiasmatic area acting upon KNDy cells to decrease kisspeptin release and thus inhibit GnRH pulses. However, several questions remain unanswered: (1) Are the coexpressed KNDy peptides, neurokinin B (NKB) and dynorphin, under seasonal regulation similar to kisspeptin? (2) Are seasonal changes in these peptides and their colocalization of D2 dopamine receptors (D2Rs) steroid dependent? and (3) Do KNDy neurons receive direct input from dopaminergic terminals? We used dual- and triple-label immunofluorescence to analyze brain sections through the ARC of ovariectomized (OVX) and OVX plus E2 ewes perfused during either the breeding season or ANS. Results showed (1) steroid-dependent and steroid-independent seasonal changes in kisspeptin and NKB, but not dynorphin, immunoreactivity; (2) increased D2R coexpression during ANS that was dependent on the presence of E2; and (3) evidence that KNDy cells receive direct contact from dopaminergic terminals and that this input increases during ANS. These results support the hypothesis that dopamine acts to inhibit GnRH secretion in ANS by directly suppressing the activity of ARC KNDy neurons, and implicate NKB as well as kisspeptin in seasonal shifts in E2-negative feedback in the sheep.

Recent evidence has implicated neurokinin B (NKB) signaling in the retrochiasmatic area (RCh) of the ewe in the LH surge. To test this hypothesis, we first lesioned NK3R neurons in this area by using a saporin conjugate (NK3-SAP). Three weeks after bilateral injection of NK3-SAP or a blank control (BLK-SAP) into the RCh, an LH surge was induced by using an artificial follicular-phase model in ovariectomized ewes. NK3-SAP lesioned approximately 88% of RCh NK3R-containing neurons and reduced the amplitude of the estrogen-induced LH surge by 58%, an inhibition similar to that seen previously with intracerebroventricular (icv) infusion of a KISS1R antagonist (p271). We next tested the hypothesis that NKB signaling in the RCh acts via kisspeptin by determining whether the combined effects of NK3R-SAP lesions and icv infusion of p271 were additive. Experiment 1 was replicated except that ewes received two sequential artificial follicular phases with infusions of p271 or vehicle using a crossover design. The combination of the two treatments decreased the peak of the LH surge by 59%, which was similar to that seen with NK3-SAP (52%) or p271 (54%) alone. In contrast, p271 infusion delayed the onset and peak of the LH surge in both NK3-SAP– and BLK-SAP–injected ewes. Based on these data, we propose that NKB signaling in the RCh increases kisspeptin levels critical for the full amplitude of the LH surge in the ewe but that kisspeptin release occurs independently of RCh input at the onset of the surge to initiate GnRH secretion.

Prenatal testosterone (T)-treated female sheep display reproductive deficits similar to women with polycystic ovarian syndrome (PCOS), including an increase in LH pulse frequency due to actions of the central GnRH pulse generator. In this study, we used multiple-label immunocytochemistry to investigate the possibility of changes in the γ-aminobutyric acid (GABA) neurotransmitter system at two key components of the GnRH pulse generator in prenatal T-treated sheep: kisspeptin/neurokinin B/dynorphin (KNDy) neurons of the arcuate nucleus, and GnRH neurons in the preoptic area (POA) and mediobasal hypothalamus (MBH). We observed a significant decrease and increase, respectively, in the number of GABAergic synapses onto POA and MBH GnRH neurons in prenatal T-treated ewes; additionally, there was a significant increase in the number of GABAergic inputs onto KNDy neurons. To determine the actions of GABA on GnRH and KNDy neurons, we examined colocalization with the chloride transporters NKCC1 and KCC2, which indicate stimulatory or inhibitory activation of neurons by GABA, respectively. Most GnRH neurons in both POA and MBH colocalized NKCC1 cotransporter whereas none contained the KCC2 cotransporter. Most KNDy neurons colocalized either NKCC1 or KCC2, and 28% of the KNDy population contained NKCC1 alone. Therefore, we suggest that, as in the mouse, GABA in the sheep is stimulatory to GnRH neurons, as well as to a subset of KNDy neurons. Increased numbers of stimulatory GABAergic inputs to both MBH GnRH and KNDy neurons in prenatal T-treated animals may contribute to alterations in steroid feedback control and increased GnRH/LH pulse frequency seen in this animal model of PCOS.

Sheep have been a useful preclinical model for the exploration of the role of kisspeptin and its receptor, KISS1R, in the control of pulsatile section of gonadotropin releasing hormone (GnRH). However, lack of reagents to interrogate KISS1R neurons in sheep has limited progress in this species. To address this limitation, we tested the feasibility of using fluorescent in situ hybridization to monitor kiss1R mRNA and a kisspeptin-saporin conjugate to ablate KISS1R-containing neurons in a cell specific manner. Using custom-designed probes for RNAscope to detect ovine kiss1R and GnRH, a high degree of colocalization was observed in the preoptic area. In future work, we can use this technique to identify mRNAs for several different possible neurotransmitters in other kiss1R-containing neurons. The effects of kiss-saporin injections was tested in mice to determine optimal concentrations and survival times. Male and female mice of two strains received infusions of 200nL of 100ng/uL, 200ng/uL and 400ng/uL concentrations of kiss-sap and blank-sap into the preoptic area, which contains the greatest density of GnRH neuron cell bodies, and, at the level of the GnRH neuron dendron prior to termination in the median eminence. Mice were perfused two or three weeks after infusion. Two adult Suffolk ewes received unilateral kiss-saporin injections (1 ul of 700 ng/ul concentration) into the preoptic area, and one ewe received kiss-sap on one side and blank-sap on the other; brains were perfused three weeks later. Mouse and sheep brains were sectioned and immunostained for GnRH. Analysis of tissue from ewes revealed that GnRH neurons were severely reduced on the side ipsilateral to the kiss-sap injection compared to the contralateral side. In contrast, GnRH neurons were found to be intact in mice, independent of injection site, dosage, survival time, strain and sex of the mice. These results indicate that kiss-sap can be used in sheep to test the physiological role of different KISS1R-containing neuronal populations. They also demonstrate a striking difference between mice and sheep in the ability to lesion GnRH cells using kiss-saporin. This raises the possibility of differences in cell surface localization and/or internalization of KISS1R between these two species, as both are needed for saporin lesions. In conclusion, these results demonstrate the feasibility of detecting kiss1R expression in sheep GnRH neurons and using GnRH cell-specific lesions, while indicating potential differences in KISS1R cellular localization between sheep and mice.

There is now general agreement that neurokinin B (NKB) acts via neurokinin-3-receptor (NK3R) to stimulate secretion of GnRH and LH in several species, including rats, mice, sheep, and humans. However, the roles of two other tachykinins, substance P (SP) and neurokinin A, which act primarily via NK1R and NK2R, respectively, are less clear. In rodents, these signaling pathways can stimulate LH release and substitute for NKB signaling; in humans, SP is colocalized with kisspeptin and NKB in the mediobasal hypothalamus. In this study, we examined the possible role of these tachykinins in control of the reproductive axis in sheep. Immunohistochemistry was used to describe the expression of SP and NK1R in the ovine diencephalon and determine whether these proteins are colocalized in kisspeptin or GnRH neurons. SP-containing cell bodies were largely confined to the arcuate nucleus, but NK1R-immunoreactivity was more widespread. However, there was very low coexpression of SP or NK1R in kisspeptin cells and none in GnRH neurons. We next determined the minimal effective dose of these three tachykinins that would stimulate LH secretion when administered into the third ventricle of ovary-intact anestrous sheep. A much lower dose of NKB (0.2 nmol) than of neurokinin A (2 nmol) or SP (10 nmol) consistently stimulated LH secretion. Moreover, the relative potency of these three neuropeptides parallels the relative selectivity of NK3R. Based on these anatomical and pharmacological data, we conclude that NKB-NK3R signaling is the primary pathway for the control of GnRH secretion by tachykinins in ewes.

The pulsatile secretion of hormones is an efficient way of coding a large variety of chemical messages. The GnRH pulse pattern determines which gonadotropin is released when and at what concentration, prescribing a detailed set of instructions to the gonads that produce changes in the steroid hormone milieu. Although GnRH neurons possess some inherent rhythmicity, they are diffusely situated within the hypothalamus and in isolation are only capable of generating physiologically irrelevant messages, hence a synchronization module exists upstream. The identity of the neural unit comprising the GnRH pulse generator is now generally thought to include KNDy neurons in the arcuate nucleus. These neurons coexpress the neuropeptides kisspeptin, neurokinin B and dynorphin A, as well as other transmitters, and are in intimate contact with the GnRH network. The GnRH pulse generator’s function is the precise control of GnRH neuron excitability, coordinated activation, stimulation of neurosecretory events, modulation of gene transcription and the mediation of the negative feedback effect of gonadal steroids. Additionally, the GnRH pulse generator is an ideal venue for the integration of various sensory and homeostatic cues that regulate reproductive functions. In this chapter we provide a historical perspective of the elegant science that sparked interest in the central mechanisms underlying the functions of the reproductive system, explain how hypotheses surrounding GnRH pulse generation have evolved and describe the current state of knowledge within the dynamic field of GnRH pulse generator research.

Work during the last decade has led to a novel hypothesis for a question that is half a century old: how is the secretory activity of GnRH neurons synchronized to produce episodic GnRH secretion. This hypothesis posits that a group of neurons in the arcuate nucleus (ARC) that contain kisspeptin, neurokinin B (NKB), and dynorphin (known as KNDy neurons) fire simultaneously to drive each GnRH pulse. Kisspeptin is proposed to be the output signal to GnRH neurons with NKB and dynorphin acting within the KNDy network to initiate and terminate each pulse, respectively. This review will focus on the importance of neuroanatomical studies in general and, more specifically, on the work of Dr Marcel Amstalden during his postdoctoral fellowship with the authors, to the development and testing of this hypothesis. Critical studies in sheep that laid the foundation for much of the KNDy hypothesis included the report that a group of neurons in the ARC contain both NKB and dynorphin and appear to form an interconnected network capable of firing synchronously, and Marcel's observations that the NKB receptor is found in most KNDy neurons, but not in any GnRH neurons. Moreover, reports that almost all dynorphin-NKB neurons and kisspeptin neurons in the ARC contained steroid receptors led directly to their common identification as "KNDy" neurons. Subsequent anatomical work demonstrating that KNDy neurons project to GnRH somas and terminals, and that kisspeptin receptors are found in GnRH, but not KNDy neurons, provided important tests of this hypothesis. Recent work has explored the time course of dynorphin release onto KNDy neurons and has begun to apply new approaches to the issue, such as RNAscope in situ hybridization and the use of whole tissue optical clearing with light-sheet microscopy. Together with other approaches, these anatomical techniques will allow continued exploration of the functions of the KNDy population and the possible role of other ARC neurons in generation of GnRH pulses.

The 3rd World Conference on Kisspeptin, "Kisspeptin 2017: Brain and Beyond" was held March 30-31 at the Rosen Centre Hotel in Orlando, Florida, providing an international forum for multidisciplinary scientists to meet and share cutting-edge research on kisspeptin biology and its relevance to human health and disease. The meeting built upon previous world conferences focused on the role of kisspeptin and associated peptides in the control of gonadotropin-releasing hormone (GnRH) secretion and reproduction. Based on recent discoveries, the scope of this meeting was expanded to include functions of kisspeptin and related peptides in other physiological systems including energy homeostasis, pregnancy, ovarian and uterine function, and thermoregulation. In addition, discussions addressed the translation of basic knowledge of kisspeptin biology to the treatment of disease, with the goal of seeking consensus about the best approaches to improve human health. The two-day meeting featured a non-traditional structure, with each day starting with poster sessions followed by lunch discussions and facilitated large-group sessions with short presentations to maximize the exchange of new, unpublished data. Topics were identified by a survey prior to the meeting, and focused on major unresolved questions, important controversies, and future directions in the field. Finally, career development activities provided mentoring for trainees and junior investigators, and networking opportunities for those individuals with established researchers in the field. Overall, the meeting was rated as a success by attendees and covered a wide range of lively and provocative discussion topics on the changing nature of the field of "kisspeptinology" and its future. This article is protected by copyright. All rights reserved.

Background: Neuronal intermediates that communicate estrogen and progesterone feedback to gonadotropin-releasing hormone (GnRH) neurons are essential for modulating reproductive cyclicity. Individually, kisspeptin and nitric oxide (NO) influence GnRH secretion. However, it is possible these 2 neuronal intermediates interact with one another to affect reproductive cyclicity. Methods: We investigated the neuroanatomical relationship of one isoform of the enzyme that synthesizes NO, neuronal NO synthase (nNOS), to kisspeptin and GnRH in adult female rhesus monkeys and sheep using dual-label immunofluorescence. Additionally, we evaluated if the phase of the reproductive cycle would affect these relationships. Results: Overall, no effect of the stage of cycle was observed for any variable in this study. In the arcuate nucleus (ARC) of sheep, 98.8 ± 3.5% of kisspeptin neurons colocalized with nNOS, and kisspeptin close-contacts were observed onto nNOS neurons. In contrast to ewes, no colocalization was observed between kisspeptin and nNOS in the infundibular ARC of primates, but kisspeptin fibers were apposed to nNOS neurons. In the preoptic area of ewes, 15.0 ± 4.2% of GnRH neurons colocalized with nNOS. In primates, 38.8 ± 10.1% of GnRH neurons in the mediobasal hypothalamus colocalized with nNOS, and GnRH close-contacts were observed onto nNOS neurons in both sheep and primates. Conclusion: Although species differences were observed, this work establishes a neuroanatomical framework between nNOS and kisspeptin and nNOS and GnRH in adult female nonhuman primates and sheep.

Puberty is a process that integrates multiple inputs ultimately resulting in an increase in gonadotrophin-releasing hormone (GnRH) secretion. Although kisspeptin neurones play an integral role in GnRH secretion and puberty onset, other systems are also likely important. One potential component is nitric oxide (NO), a gaseous neurotransmitter synthesised by nitric oxide synthase (NOS). The present study aimed to neuroanatomically characterise neuronal NOS (nNOS) in prepubertal female sheep and determine whether oestradiol exerts effects on this system. Luteinising hormone secretion was reduced by oestradiol treatment in prepubertal ovariectomised ewes. Neurones immunoreactive for nNOS were identified in several areas, with the greatest number present in the ventrolateral portion of the ventromedial hypothalamus, followed by the ventromedial hypothalamus, preoptic area (POA) and arcuate nucleus (ARC). Next, we determined whether nNOS neurones contained oestrogen receptor (ER)α and could potentially communicate oestradiol (E2 ) feedback to GnRH neurones. Neuronal NOS neurones contained ERα with the percentage of coexpression (12%-40%) depending upon the area analysed. We next investigated whether a neuroanatomical relationship existed between nNOS and kisspeptin or nNOS and GnRH neurones. A high percentage of kisspeptin neurones in the POA (79%) and ARC (98%) colocalised with nNOS. Kisspeptin close contacts were also associated with nNOS neurones. A greater number of close contacts were observed in the ARC than the POA. A high percentage of POA GnRH neurones (79%) also expressed nNOS, although no GnRH close contacts were observed onto nNOS neurones. Neither the numbers of nNOS neurones in the POA or hypothalamus, nor the percentage of nNOS coexpression with GnRH, kisspeptin or ERα were influenced by oestradiol. These experiments reveal that a neuroanatomical relationship exists between both nNOS and kisspeptin and nNOS and GnRH in prepubertal ewes. Therefore, nNOS may act both directly and indirectly to influence GnRH secretion in prepubertal sheep.

Work over the last 15 years on the control of pulsatile LH secretion has focused largely on a set of neurons in the arcuate nucleus (ARC) that contains two stimulatory neuropeptides, critical for fertility in humans (kisspeptin and neurokinin B (NKB)) and the inhibitory endogenous opioid peptide (EOP), dynorphin, and are now known as KNDy (kisspeptin-NKB-dynorphin) neurons. In this review, we consider the role of each of the KNDy peptides in the generation of GnRH pulses and the negative feedback actions of ovarian steroids, with an emphasis on NKB and dynorphin. With regard to negative feedback, there appear to be important species differences. In sheep, progesterone inhibits GnRH pulse frequency by stimulating dynorphin release, and estradiol inhibits pulse amplitude by suppressing kisspeptin. In rodents, the role of KNDy neurons in estrogen negative feedback remains controversial, progesterone may inhibit GnRH via dynorphin, but the physiological significance of this action is unclear. In primates, an EOP, probably dynorphin, mediates progesterone negative feedback, and estrogen inhibits kisspeptin expression. In contrast, there is now compelling evidence from several species that kisspeptin is the output signal from KNDy neurons that drives GnRH release during a pulse and may also act within the KNDy network to affect pulse frequency. NKB is thought to act within this network to initiate each pulse, although there is some redundancy in tachykinin signaling in rodents. In ruminants, dynorphin terminates GnRH secretion at the end of pulse, most likely acting on both KNDy and GnRH neurons, but the data on the role of this EOP in rodents are conflicting.

Problem The chemical propanil enhances antibody responses to a heat-killed Streptococcus pneumoniae (HKSP) vaccine. The enhanced response is dependent on gonads in females, but independent of gonads in males. The sex differences in the immune response may be due to sexual differentiation of the immune system or sex chromosome complement. Method of study To test the hypothesis that the immune system is sexually differentiated, newborn C57BL/6 pups were treated with testosterone propionate (TP) or placebo. The role of sex chromosome complement was investigated using the 4-core genotypes (FCG) model of XXF and XYF gonadal females (ovaries), and XXM and XYM gonadal males (testes). For some experiments, mice were gonadectomized or sham gonadectomized. All mice were vaccinated with HKSP, treated with propanil, and the antibody response determined at day seven. Results Neonatal TP did not alter the response to HKSP. In FCG mice, propanil significantly enhanced the immune response in XXF females and XXM males, but not in XYF females or XYM males. Conclusion The immune system of females was not masculinized by neonatal TP treatment. Sex chromosome complement significantly contributes to the sexually dimorphic immune response after propanil exposure.

Two modes of gonadotropin-releasing hormone (GnRH) and luteinizing hormone (LH) secretion are necessary for female fertility: surge and episodic secretion. However, the neural systems that regulate these GnRH secretion patterns are still under investigation. The neuropeptide somatostatin (SST) inhibits episodic LH secretion in humans and sheep, and several lines of evidence suggest SST may regulate secretion during the LH surge. In this study, we examined whether SST alters the LH surge in ewes by administering a SST receptor (SSTR) 2 agonist (octreotide) or antagonist [CYN154806 (CYN)] into the third ventricle during an estrogen-induced LH surge and whether endogenous SST alters episodic LH secretion. Neither octreotide nor CYN altered the amplitude or timing of the LH surge. Administration of CYN to intact ewes during the breeding season or anestrus increased LH secretion and increased c-Fos in a subset GnRH and kisspeptin cells during anestrus. To determine if these stimulatory effects are steroid dependent or independent, we administered CYN to ovariectomized ewes. This SSTR2 antagonist increased LH pulse frequency in ovariectomized ewes during anestrus but not during the breeding season. This study provides evidence that endogenous SST contributes to the control of LH secretion. The results demonstrate that SST, acting through SSTR2, inhibits episodic LH secretion, likely acting in the mediobasal hypothalamus, but action at this receptor does not alter surge secretion. Additionally, these data provide evidence that SST contributes to the steroid-independent suppression of LH pulse frequency during anestrus.

Neurokinin B (NKB) is essential for human reproduction and has been shown to stimulate luteinising hormone (LH) secretion in several species, including sheep. Ewes express the neurokinin-3 receptor (NK3R) in the retrochiasmatic area (RCh) and there is one report that placement of senktide, an NK3R agonist, therein stimulates LH secretion that resembles an LH surge in ewes. In the present study, we first confirmed that local administration of senktide to the RCh produced a surge-like increase in LH secretion, and then tested the effects of this agonist in two other areas implicated in the control of LH secretion and where NK3R is found in high abundance: the preoptic area (POA) and arcuate nucleus (ARC). Bilateral microimplants containing senktide induced a dramatic surge-like increase in LH when given in the POA similar to that seen with RCh treatment. By contrast, senktide treatment in the ARC resulted in a much smaller but significant increase in LH concentrations suggestive of an effect on tonic secretion. The possible role of POA and RCh NK3R activation in the LH surge was next tested by treating ewes with SB222200, an NK3R antagonist, in each area during an oestradiol-induced LH surge. SB222200 in the RCh, but not in the POA, reduced the LH surge amplitude by approximately 40% compared to controls, indicating that NK3R activation in the former region is essential for full expression of the pre-ovulatory LH surge. Based on these data, we propose that the actions of NKB in the RCh are an important component of the pre-ovulatory LH surge in ewes.

The recent description of infertility in humans with loss-of-function mutations in genes for neurokinin B (NKB) or its receptor (NK3R) has focused attention on the importance of this tachykinin in the control of GnRH secretion. In a number of species, NKB neurons in the arcuate nucleus also produce two other neuropeptides implicated in the control of GnRH secretion: (1) kisspeptin, which is also essential for fertility in humans, and (2) dynorphin, an inhibitory endogenous opioid peptide. A number of characteristics of this neuronal population led to the hypothesis that they may be responsible for driving synchronous release of GnRH during episodic secretion of this hormone, and there is now considerable evidence to support this hypothesis in sheep and goats. In this article, we briefly review the history of work on the NKB system in sheep and then review the anatomy of NKB signaling in the ewe. We next describe evidence from a number of species that led to development of a model for the role of these neurons in episodic GnRH secretion. Finally, we discuss recent experiments in sheep and goats that tested this hypothesis and led to a modified version of the model, and then broaden our focus to briefly consider the possible roles of NKB in other species and systems.

The neuropeptides neurokinin B (NKB) and kisspeptin are potent stimulators of gonadotrophin-releasing hormone (GnRH)/luteinsing hormone (LH) secretion and are essential for human fertility. We have recently demonstrated that selective activation of NKB receptors (NK3R) within the retrochiasmatic area (RCh) and the preoptic area (POA) triggers surge-like LH secretion in ovary-intact ewes, whereas blockade of RCh NK3R suppresses oestradiol-induced LH surges in ovariectomised ewes. Although these data suggest that NKB signalling within these regions of the hypothalamus mediates the positive-feedback effects of oestradiol on LH secretion, the pathway through which it stimulates GnRH/LH secretion remains unclear. We proposed that the action of NKB on RCh neurones drives the LH surge by stimulating kisspeptin-induced GnRH secretion. To test this hypothesis, we quantified the activation of the preoptic/hypothalamic populations of kisspeptin neurones in response to POA or RCh administration of senktide by dual-label immunohistochemical detection of kisspeptin and c-Fos (i.e. marker of neuronal activation). We then administered the NK3R agonist, senktide, into the RCh of ewes in the follicular phase of the oestrous cycle and conducted frequent blood sampling during intracerebroventricular infusion of the kisspeptin receptor antagonist Kp-271 or saline. Our results show that the surge-like secretion of LH induced by RCh senktide administration coincided with a dramatic increase in c-Fos expression within arcuate nucleus (ARC) kisspeptin neurones, and was completely blocked by Kp-271 infusion. We substantiate these data with evidence of direct projections of RCh neurones to ARC kisspeptin neurones. Thus, NKB-responsive neurones in the RCh act to stimulate GnRH secretion by inducing kisspeptin release from KNDy neurones.

Puberty onset involves increased gonadotrophin-release (GnRH) release as a result of decreased sensitivity to oestrogen (E2 )-negative feedback. Because GnRH neurones lack E2 receptor α, this pathway must contain interneurones. One likely candidate is KNDy neurones (kisspeptin, neurokinin B, dynorphin). The overarching hypothesis of the present study was that the prepubertal hiatus in luteinising hormone (LH) release involves reduced kisspeptin and/or heightened dynorphin input. We first tested the specific hypothesis that E2 would reduce kisspeptin-immunopositive cell numbers and increase dynorphin-immunopositive cell numbers. We found that kisspeptin cell numbers were higher in ovariectomised (OVX) lambs than OVX lambs treated with E2 (OVX+ E2 ) or those left ovary-intact. Very few arcuate dynorphin cells were identified in any group. Next, we hypothesised that central blockade of κ-opioid receptor (KOR) would increase LH secretion at a prepubertal (6 months) but not postpubertal (10 months) age. Luteinising hormone pulse frequency and mean LH increased during infusion of a KOR antagonist, norbinaltorphimine, in OVX + E2 lambs at the prepubertal age but not in the same lambs at the postpubertal age. We next hypothesised that E2 would increase KOR expression in GnRH neurones or alter synaptic input to KNDy neurones in prepubertal ewes. Oestrogen treatment decreased the percentage of GnRH neurones coexpressing KOR (approximately 68%) compared to OVX alone (approximately 78%). No significant differences in synaptic contacts per cell between OVX and OVX + E2 groups were observed. Although these initial data are consistent with dynorphin inhibiting pulsatile LH release prepubertally, additional work will be necessary to define the source and mechanisms of this inhibition.

This review focuses on the importance of cortisol in mediating the inhibitory effects of psychosocial stress on reproduction in females. In particular, we have summarized our research in sheep where we have systematically established whether cortisol is both sufficient and necessary to suppress reproductive hormone secretion and inhibit sexual behaviour. Our findings are put into context with previous work and are used to develop important concepts as well as to identify productive further lines of investigation. It is clear that cortisol is necessary to inhibit some, but not all, aspects of reproduction in female sheep. These actions vary with reproductive state, and there are important interactions with gonadal steroids. The impact of cortisol on the tonic secretion of gonadotrophin-releasing hormone and luteinizing hormone has been investigated extensively, but less is known about the surge secretion of these hormones and their effects on sexual behaviour. Furthermore, there are separate effects of cortisol in the brain (hypothalamus) and at the anterior pituitary, illustrating that there are different mechanisms of action. Thus, although cortisol is important in mediating some of the effects of stress on reproduction, we need to look beyond cortisol and investigate some of the other mechanisms and mediators that relay the effects of stress on reproduction. In this regard, we propose that a group of neurons in the hypothalamus that co-synthesize kisspeptin, neurokinin B and dynorphin, termed KNDy cells, play important roles in mediating the effects of cortisol on reproduction. This hypothesis needs to be rigorously tested.

Photoperiod determines the timing of reproductive activity in many species, yet the neural pathways whereby day length is transduced to a signal influencing gonadotropin-releasing hormone (GnRH) release are not fully understood. Physical lesions of the lateral preoptic area (lPOA)/rostral anterior hypothalamic area (rAHA) in female sheep extend the period of estrous cyclicity during inhibitory photoperiods. In the present study we sought to determine whether destroying only neurons and not fibers of passage in this area would lead to similar resistance to photosuppression. Additionally, neural tract-tracing was used to map connectivity between the lPOA/rAHA and other hypothalamic areas implicated in photoperiodic regulation of reproduction. Progesterone secretion was monitored in six sheep to determine estrous cycles for 90 days during a short-day (permissive) photoperiod. Three sheep then received bilateral injections of the excitotoxic glutamate analog, n-methyl-aspartic acid, directed toward the lPOA/rAHA, whereas three others served as controls. All were then exposed to a long-day (suppressive) photoperiod for 120 days. Control sheep ceased cycling at 40 ± 10 days (mean ± SEM), whereas lesioned sheep continued cycling through the end of the study. The results of the tract-tracing study revealed both afferent and efferent projections to the medial POA, retrochiasmatic area, arcuate nucleus, and premammillary region. Furthermore, close proximal associations with GnRH neurons from efferent projections were observed. We conclude that neurons located within the lPOA/rAHA are important for timing cessation of estrous cycles during photosuppression and that this area communicates directly with GnRH neurons and other hypothalamic areas involved in the photoperiodic regulation of reproduction.

There is now considerable evidence that dynorphin neurons mediate the negative feedback actions of progesterone to inhibit GnRH and LH pulse frequency, but the specific neurons have yet to be identified. In ewes, dynorphin neurons in the arcuate nucleus (ARC) and preoptic area (POA) are likely candidates based on colocalization with progesterone receptors. These studies tested the hypothesis that progesterone negative feedback occurs in either the ARC or POA by determining whether microimplants of progesterone into either site would inhibit LH pulse frequency (study 1) and whether microimplants of the progesterone receptor antagonist, RU486, would disrupt the inhibitory effects of peripheral progesterone (study 2). Both studies were done in ovariectomized (OVX) and estradiol-treated OVX ewes. In study 1, no inhibitory effects of progesterone were observed during treatment in either area. In study 2, microimplants of RU486 into the ARC disrupted the negative-feedback actions of peripheral progesterone treatments on LH pulse frequency in both OVX and OVX+estradiol ewes. In contrast, microimplants of RU486 into the POA had no effect on the ability of systemic progesterone to inhibit LH pulse frequency. We thus conclude that the ARC is one important site of progesterone-negative feedback in the ewe. These data, which are the first evidence on the neural sites in which progesterone inhibits GnRH pulse frequency in any species, are consistent with the hypothesis that ARC dynorphin neurons mediate this action of progesterone.

Seasonal reproduction represents a naturally occurring example of functional plasticity in the adult brain as it reflects changes in neuroendocrine pathways controlling gonadotropin-releasing hormone (GnRH) secretion and, in particular, the responsiveness of GnRH neurons to estradiol negative feedback. Structural plasticity within this neural circuitry may, in part, be responsible for seasonal switches in the negative feedback control of GnRH secretion that underlie annual reproductive transitions. We review evidence for structural changes in the circuitry responsible for seasonal inhibition of GnRH secretion in sheep. These include changes in synaptic inputs onto GnRH neurons, as well as onto dopamine neurons in the A15 cell group, a nucleus that plays a key role in estradiol negative feedback. We also present preliminary data suggesting a role for neurotrophins and neurotrophin receptors as an early mechanistic step in the plasticity that accompanies seasonal reproductive transitions in sheep. Finally, we review recent evidence suggesting that kisspeptin cells of the arcuate nucleus constitute a critical intermediary in the control of seasonal reproduction. Although a majority of the data for a role of neuronal plasticity in seasonal reproduction has come from the sheep model, the players and principles are likely to have relevance for reproduction in a wide variety of vertebrates, including humans, and in both health and disease.

We compare long-term outcomes in patients with node negative early stage breast cancer treated with breast radiotherapy (RT) without the axillary RT field after sentinel lymph node dissection (SLND) or axillary lymph node dissection (ALND). We hypothesize that though tangential RT was delivered to the breast tissue, it at least partially sterilized occult axillary nodal metastases thus providing low nodal failure rates. Between 1995 and 2001, 265 patients with AJCC stages I–II breast cancer were treated with lumpectomy and either SLND (cohort SLND) or SLND and ALND (cohort ALND). Median follow-up was 9.9 years (range 8.3–15.3 years). RT was administered to the whole breast to the median dose of 48.2 Gy (range 46.0–50.4 Gy) plus boost without axillary RT. Chi-square tests were employed in comparing outcomes of two groups for axillary and supraclavicular failure rates, ipsilateral in-breast tumor recurrence (IBTR), distant metastases (DM), and chronic complications. Progression-free survival (PFS) was compared using log-rank test. There were 136/265 (51%) and 129/265 (49%) patients in the SLND and ALND cohorts, respectively. The median number of axillary lymph nodes assessed was 2 (range 1–5) in cohort SLND and 18 (range 7–36) in cohort ALND (P < 0.0001). Incidence of AFR and SFR in both cohorts was 0%. The rates of IBTR and DM in both cohorts were not significantly different. Median PFS in the SLND cohort is 14.6 years and 10-year PFS is 88.2%. Median PFS in the ALND group is 15.0 years and 10-year PFS is 85.7%. At a 10-year follow-up chronic lymphedema occurred in 5/108 (4.6%) and 40/115 (34.8%) in cohorts SLND and ALND, respectively (P = 0.0001). This study provides mature evidence that patients with negative nodes, treated with tangential breast RT and SLND alone, experience low AFR or SFR. Our findings, while awaiting mature long-term data from NSABP B-32, support that in patients with negative axillary nodal status such treatment provides excellent long-term cure rates while avoiding morbidities associated with ALND or addition of axillary RT field.

Recent evidence has implicated neurokinin B (NKB) in the complex neuronal network mediating the effects of gonadal steroids on the regulation of gonadotrophin-releasing hormone (GnRH) secretion. Because the neurokinin 3 receptor (NK3R) is considered to mediate the effects of NKB at the cellular level, we determined the distribution of immunoreactive NK3R in the septal region, preoptic area (POA) and hypothalamus of the ewe. NK3R cells and/or fibres were found in areas including the bed nucleus of the stria terminalis, POA, anterior hypothalamic and perifornical areas, dopaminergic A15 region, dorsomedial and lateral hypothalamus, arcuate nucleus (ARC) and the ventral premammillary nucleus. We also used dual-label immunocytochemistry to determine whether a neuroanatomical basis for direct modulation of GnRH neurones by NKB was evident. No GnRH neurones at any rostral-caudal level were observed to contain NK3R immunoreactivity, although GnRH neurones and fibres were in proximity to NK3R-containing fibres. Because NKB fibres formed close contacts with NKB neurones in the ARC, we determined whether these NKB neurones also contained immunoreactive NK3R. In luteal-phase ewes, 64% +/- 11 of NKB neurones colocalised NK3R. In summary, NK3R is distributed in areas of the sheep POA and hypothalamus known to be involved in the control of reproductive neuroendocrine function. Colocalisation of NK3R in NKB neurones of the ARC suggests a potential mechanism for the autoregulation of this subpopulation; however, the lack of NK3R in GnRH neurones suggests that the actions of NKB on GnRH neurosecretory activity in the ewe are mediated indirectly via other neurones and/or neuropeptides.

International audience; Sheep are seasonal breeders, experiencing a period of reproductive quiescence during spring and early summer. During the non-breeding period, kisspeptin expression in the arcuate nucleus is markedly reduced. This strongly suggests that the mechanisms that control seasonal changes in reproductive function involve kisspeptin neurons. Kisspeptin cells appear to regulate GnRH neurons and transmit sex-steroid feedback to the reproductive axis. Since the non-breeding season is characterized by increased negative feedback of estrogen on GnRH secretion, the kisspeptin neurons seem to be fundamentally involved in the determination of breeding state. The reduction in kisspeptin neuronal function during the non-breeding season can be corrected by infusion of kisspeptin, which causes ovulation in seasonally acyclic females. (C) 2008 Elsevier Inc. All rights reserved.

Reproductive activity in sheep is seasonal, being activated by short-day photoperiods and inhibited by long days. During the nonbreeding season, GnRH secretion is reduced by both steroid-independent and steroid-dependent (increased response to estradiol negative feedback) effects of photoperiod. Kisspeptin (also known as metastin) and gonadotropin-inhibitory hormone (GnIH, or RFRP) are two RFamide neuropeptides that appear critical in the regulation of the reproductive neuroendocrine axis. We hypothesized that expression of kisspeptin and/or RFRP underlies the seasonal change in GnRH secretion. We examined kisspeptin and RFRP (protein and mRNA) expression in the brains of ovariectomized (OVX) ewes treated with estradiol (OVX+E) during the nonbreeding and breeding seasons. In OVX+E ewes, greater expression of kisspeptin and Kiss1 mRNA in the arcuate nucleus and lesser expression of RFRP (protein) in the dorsomedial nucleus of the hypothalamus were concurrent with the breeding season. There was also a greater number of kisspeptin terminal contacts onto GnRH neurons and less RFRP-GnRH contacts during the breeding season (compared with the nonbreeding season) in OVX+E ewes. Comparison of OVX and OVX+E ewes in the breeding and nonbreeding season revealed a greater effect of steroid replacement on inhibition of kisspeptin protein and Kiss1 mRNA expression during the nonbreeding season. Overall, we propose that the two RFamide peptides, kisspeptin and RFRP, act in concert, with opposing effects, to regulate the activity of GnRH neurons across the seasons, leading to the annual change in fertility and the cyclical seasonal transition from nonbreeding to breeding season.

Kisspeptin is a potent stimulator of GnRH secretion that has been implicated in the feedback actions of ovarian steroids. In ewes, the majority of hypothalamic kisspeptin neurons are found in the arcuate nucleus (ARC), with a smaller population located in the preoptic area. Most arcuate kisspeptin neurons express estrogen receptor-α, as do a set of arcuate neurons that contain both dynorphin and neurokinin B (NKB), suggesting that all three neuropeptides are colocalized in the same cells. In this study we tested this hypothesis using dual immunocytochemistry and also determined if kisspeptin neurons contain MSH or agouti-related peptide. To assess colocalization of kisspeptin and dynorphin, we used paraformaldehyde-fixed tissue from estrogen-treated ovariectomized ewes in the breeding season (n = 5). Almost all ARC, but no preoptic area, kisspeptin neurons contained dynorphin. Similarly, almost all ARC dynorphin neurons contained kisspeptin. In experiment 2 we examined colocalization of kisspeptin and NKB in picric-acid fixed tissue collected from ovary intact ewes (n = 9). Over three quarters of ARC kisspeptin neurons also expressed NKB, and a similar percentage of NKB neurons contained kisspeptin. In contrast, no kisspeptin neurons stained for MSH or agouti-related peptide. These data demonstrate that, in the ewe, a high percentage of ARC kisspeptin neurons also produce dynorphin and NKB, and we propose that a single subpopulation of ARC neurons contains all three neuropeptides. Because virtually all of these neurons express estrogen and progesterone re-ceptors, they are likely to relay the feedback effects of these steroids to GnRH neurons to regulate reproductive function.

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 [Arg14, Lys15] 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.

Seasonal anoestrus in the ewe results from enhanced oestrogen negative feedback. Recent data have implicated the ventromedial preoptic area (vmPOA) as an important site of oestrogen action. This study addressed whether NO acts within the vmPOA to inhibit LH during seasonal anoestrus. In Experiment 1, microimplants containingNω-nitro-l-arginine methyl ester (l-NAME, NOS inhibitor),S-methyl thiocitrulline (SMTC, neural NOS (nNOS) inhibitor) or empty implants (control) were administered during mid-anoestrus to the vmPOA.l-NAME, but not SMTC, significantly increased LH pulse frequency. For Experiment 2, ewes in late anoestrus were administered 7-nitroindazole (7NI; nNOS inhibitor),l-NAME, SMTC, or empty implants. 7NI, but notl-NAME or SMTC, increased LH pulse frequency. In Experiment 3, the effects of microimplants and microinjections ofl-NAME were compared in mid-anoestrus. Microinjections ofl-NAME (300 nl at 10 μg/μl) increased LH pulse frequency, but microimplants did not. In late anoestrus, similar microinjections were ineffective. Taken together, the results of Experiments 1–3 suggested that NO inhibition may be stronger during the middle than at the end of seasonal anoestrus. To test this hypothesis, ewes in Experiment 4 received microinjection ofl-NAME or vehicle thrice during the non-breeding season; none of the treatments increased LH pulse frequency. These results indicate that NO plays a role in the vmPOA in suppressing LH secretion during seasonal anoestrus because NOS inhibitors were consistently stimulatory when LH pulse frequency was low. However, the inconsistent and modest effects of these inhibitors suggest that NO actions in this area cannot completely account for the effects of inhibitory photoperiod.

Kisspeptin neurones located in the arcuate nucleus (ARC) and preoptic area (POA) are critical mediators of gonadal steroid feedback onto gonadotrophin-releasing hormone (GnRH) neurones. ARC kisspeptin cells that co-localise neurokinin B (NKB) and dynorphin (Dyn), are collectively referred to as KNDy (Kisspeptin/NKB/Dyn) neurones, and have been shown in mice to also co-express the vesicular glutamate transporter, vGlut2, an established glutamatergic marker. The ARC in rodents has long been known as a site of hormone-induced neuroplasticity, and changes in synaptic inputs to ARC neurones in rodents occur over the oestrous cycle. Based on this evidence, the the present study aimed to examine possible changes across the ovine oestrous cycle in synaptic inputs onto kisspeptin cells in the ARC (KNDy) and POA, and inputs onto GnRH neurones. Gonadal-intact breeding season ewes were perfused using 4% paraformaldehyde during either the luteal or follicular phase of the oestrous cycle, with the latter group killed at the time of the luteinising hormone (LH) surge. Hypothalamic sections were processed for triple-label immunodetection of kisspeptin/vGlut2/synaptophysin or kisspeptin/vGlut2/GnRH. The total numbers of synaptophysin- and vGlut2-positive inputs to ARC KNDy neurones were significantly increased at the time of the LH surge compared to the luteal phase; because these did not contain kisspeptin, they do not arise from KNDy neurones. By contrast to the ARC, the total number of synaptophysin-positive inputs onto POA kisspeptin neurones did not differ between luteal phase and surge animals. The total number of kisspeptin and vGlut2 inputs onto GnRH neurones in the mediobasal hypothalamus (MBH) was also increased during the LH surge, and could be attributed to an increase in the number of KNDy (double-labelled kisspeptin + vGlut2) inputs. Taken together, these results provide novel evidence of synaptic plasticity at the level of inputs onto KNDy and GnRH neurones during the ovine oestrous cycle. Such changes may contribute to the generation of the preovulatory GnRH/LH surge.

Prenatal testosterone (T)-treated ewes display a constellation of reproductive defects that closely mirror those seen in PCOS women, including altered hormonal feedback control of GnRH. Kisspeptin/neurokinin B/dynorphin (KNDy) neurons of the arcuate nucleus (ARC) play a key role in steroid feedback control of GnRH secretion, and prenatal T treatment in sheep causes an imbalance of KNDy peptide expression within the ARC. In the present study, we tested the hypothesis that prenatal T exposure, in addition to altering KNDy peptides, leads to changes in the morphology and synaptic inputs of this population, kisspeptin cells of the preoptic area (POA), and GnRH cells. Prenatal T treatment significantly increased the size of KNDy cell somas, whereas POA kisspeptin, GnRH, agouti-related peptide, and proopiomelanocortin neurons were each unchanged in size. Prenatal T treatment also significantly reduced the total number of synaptic inputs onto KNDy neurons and POA kisspeptin neurons; for KNDy neurons, the decrease was partly due to a decrease in KNDy-KNDy synapses, whereas KNDy inputs to POA kisspeptin cells were unaltered. Finally, prenatal T reduced the total number of inputs to GnRH cells in both the POA and medial basal hypothalamus, and this change was in part due to a decreased number of inputs from KNDy neurons. The hypertrophy of KNDy cells in prenatal T sheep resembles that seen in ARC kisspeptin cells of postmenopausal women, and together with changes in their synaptic inputs and projections to GnRH neurons, may contribute to defects in steroidal control of GnRH observed in this animal model.

In this chapter, I describe the similarities and differences among species in the neuroendocrine control of gonadotropin secretion. The discussion focuses on mice and rats, sheep, and primates because considerable data are available on these mammals, and information from reflex ovulators, guinea pigs, and other domestic animals is also included. I first describe species differences in ovarian cycles and review the development of current concepts for the feedback relationships between the ovary and hypothalamo–hypophysial unit underlying these cycles. I then consider species differences in the feedback control of the luteinizing hormone (LH) surge, tonic LH secretion, and follicle-stimulating hormone secretion. Next I present an overview of the anatomical areas necessary for tonic and surge secretion of LH, the sites of steroid feedback, and the roles of norepinephrine, endogenous opioid peptides, and kisspeptin. Finally, the mechanisms responsible for the physiological suppression of reproduction that occurs prior to puberty, during lactation and periods of undernutrition, in response to social stimuli stressors, and in seasonal breeders are briefly compared among mammals.

Reproduction in the ewe is dominated by two distinct rhythms: a 16–17-day estrous cycle and an annual cycle of fertility. Events of the estrous cycle are controlled by the interactions of several hormones (gonadotropin-releasing hormone from the hypothalamus; luteinizing hormone and follicle-stimulating hormone from the pituitary; estradiol, inhibin, and progesterone from the ovary; and prostaglandin F 2α from the uterus), while seasonal reproductive patterns are determined by melatonin from the pineal gland. In this chapter, we will first describe the behavioral and hormonal patterns during the cycle, and then consider the mechanisms controlling estrous behavior and the secretion of each hormone. We will next synthesize these components to provide an integrated model for the ovarian cycle. Finally, we will discuss the mechanisms underlying the annual breeding and anestrous seasons, and how pheromones and social cues act in anestrus to re-initiate fertility. Throughout, we will emphasize new data obtained in the last decade, while integrating this information with established concepts described in the previous version of this chapter.

GABA has been shown to play an important role in the control of gonadotropin-releasing hormone (GnRH) and luteinizing hormone secretion in many mammals. In sheep, seasonal differences in the ability of GABA-B receptor antagonists to alter pulsatile luteinizing hormone secretion have led to the hypothesis that this receptor subtype mediates the increased inhibitory effects of estradiol on GnRH and luteinizing hormone pulse frequency seen during the non-breeding season (anestrus). The aim of the present study was to use multiple-label immunocytochemistry to determine if ovine GnRH neurons contain the GABA-B receptor subunits R1 and/or R2, and to determine whether there are seasonal differences in the colocalization of these subunits in GnRH neurons. A majority of GnRH cells in the preoptic area, anterior hypothalamic area, and medial basal hypothalamus of both breeding season and anestrous ewes contained either GABA-B R1 or R2 subunits; a subset of GnRH neurons in breeding season (42%) and anestrous ewes (60%) contained both subunits. In contrast to colocalization within cell bodies, GnRH fibers in the median eminence did not colocalize GABA-B receptor subunits. Although the percentage of GnRH neurons expressing GABA-B receptor subunits tended to be higher in anestrus than in the breeding season, there were no significant seasonal differences in R1 and R2 subunit colocalization in GnRH cell bodies. Thus, while GABA may act directly on GnRH cell bodies via GABA-B receptors in the sheep, any role that GABA-B receptors may play in seasonal reproductive changes is likely mediated by other neurons afferent to GnRH cells.

The ability of steroids to inhibit LH secretion is enhanced during undernutrition. To identify potential hypothalamic sites at which this enhancement may occur, we examined LH secretion in feed-restricted or fed young wethers treated with locally administered metabolites of testosterone. In experiment 1, microimplants containing crystalline estradiol-17beta (E) or cholesterol were administered via chronic guide tubes directed to the preoptic area (POA) or ventromedial hypothalamus (VMH) in fed or feed-restricted wethers. E treatment in the VMH decreased LH pulse frequency, pulse amplitude, and mean LH concentration in feed-restricted, but not fed, wethers. E may act in the POA to suppress LH under feed restriction, but definite conclusions cannot be drawn because of steroid-independent effects of feed restriction on LH pulse frequency. In experiment 2, the effect of dihydrotestosterone (DHT) in the VMH was determined. DHT administration to the VMH did not alter LH secretion in either feed-restricted or fed wethers. Thus the VMH is one site wherein E negative feedback is enhanced during feed restriction in the wether. In contrast, we found no evidence for enhanced responsiveness to androgen negative feedback within the VMH of feed-restricted wethers. We suggest that increased sensitivity within the VMH to E, but not to DHT, is important for suppressing LH secretion in undernourished male sheep.

Recent studies suggest that the endogenous opioid peptide, dynorphin, is an important mediator of progesterone negative feedback on GnRH pulse frequency in the ewe. These experiments tested this hypothesis by examining the effects of progesterone on dynorphin A concentrations in cerebrospinal fluid (CSF) collected from the third ventricle and expression of preprodynorphin (PPD) mRNA in hypothalamic nuclei. CSF was collected every 10 min for 5 h in three groups of ewes: 1) ovary-intact ewes during the luteal phase (d 6-7 of estrous cycle); 2) ewes 6-7 d after ovariectomy (OVX); and 3) OVX ewes treated for 6-7 d with implants that produced luteal-phase progesterone levels. Diencephalic tissue from these ewes was then collected and processed for in situ hybridization using an ovine cDNA probe against PPD. Progesterone treatment increased dynorphin A concentrations in CSF over that observed in untreated OVX ewes; CSF dynorphin A concentrations in ovary-intact ewes were midway between the other groups. OVX significantly decreased the number of PPD mRNA-expressing cells in the preoptic area (POA), anterior hypothalamic area (AHA), and arcuate nucleus (ARC), with no change seen in any other PPD-expressing nuclei. Progesterone treatment of OVX ewes restored PPD expression in the POA and AHA to levels seen in luteal-phase animals but had no effect on PPD expression in the ARC. These results are consistent with the hypothesis that progesterone acts via dynorphin neurons to inhibit pulsatile GnRH secretion and point to dynorphin neurons in the POA, AHA, and ARC as potential mediators of this action during the luteal phase.

Endogenous opioid peptides (EOP) are important modulators in a variety of neuroendocrine systems, including those mediating reproduction, energy balance, lactation, and stress. Recent work in the ewe has implicated the EOP, dynorphin (DYN), in the inhibitory effects of progesterone on pulsatile gonadotropin releasing hormone secretion. Although DYN is involved in a number of hypothalamic functions in the sheep, little is known regarding the localization of preprodynorphin (PPD) expression and its major product DYN A (1–17). In this study, we determined the distribution of PPD mRNA and DYN A-containing cell bodies in the brains of ovary-intact, luteal ewes. To detect PPD mRNA, an ovine PPD mRNA was subcloned by reverse transcription–polymerase chain reaction from sheep hypothalamus and used to create a 35 S-labeled riboprobe for in situ hybridization. Neurons that expressed PPD mRNA and DYN A immunoreactivity were widely distributed in the ovine preoptic area and hypothalamus. PPD mRNA-expressing cells were seen in the supraoptic nucleus, paraventricular nucleus, preoptic area, anterior hypothalamus area, bed nucleus of the stria terminalis, ventromedial nucleus (VMN), dorsomedial nucleus of the hypothalamus, and the arcuate nucleus. All of these regions also contained DYN A-positive cell bodies except for the VMN, raising the possibility that PPD is preferentially processed into other peptide products in the VMN. In summary, based on the expression of both mRNA and peptide, DYN cells are located in a number of key hypothalamic regions involved in the neuroendocrine control of homeostasis in sheep.

Thyroid hormones are permissive for various species to enter seasonal anestrus. In the ewe they act centrally to permit the onset of potent estradiol-negative feedback responsible for anestrus, but the specific sites of action are unknown. Therefore, we tested whether T(4) replacement via chronic microimplants in any of five brain areas could reverse the reproductive effects of thyroidectomy. Diffusion of (125)I-T(4) from the microimplant was largely (98%) limited to a 1.2-mm radius. A marked decline in LH concentration in ovariectomized, estradiol-treated ewes was used as an index for anestrus. In experiment 1, all thyroidectomized (THX) ewes with microimplants in the medial preoptic area, A15 area, and medial basal hypothalamus failed to enter anestrus; instead, LH levels remained elevated, similar to those in untreated THX controls. In ventromedial preoptic area (vmPOA)-microimplanted ewes, only the two animals with the most caudal microimplants entered anestrus, as did thyroid-intact controls and THX ewes receiving icv or sc T(4) replacement. In experiment 2, all vmPOA-treated ewes with similar placements to those effective in experiment 1 along with all ewes microimplanted in the premammillary region entered neuroendocrine anestrus. Thus, the premammillary region and vmPOA are brain sites in which thyroid hormones act to permit the onset of seasonal anestrus.

In ewes, anestrus results from a reduction in LH pulsatility due to an increased sensitivity of the hypothalamic estradiol negative feedback system. Considerable evidence has implicated the A15 group of dopaminergic neurons in the retrochiasmatic area in this seasonally dependent estradiol effect. Moreover, estradiol administered to the retrochiasmatic area in ovariectomized anestrous ewes inhibits LH secretion. However, A15 neurons do not appear to contain the classical estrogen receptors (ERα). Therefore, we tested the hypothesis that β-estrogen receptors mediate the action of estradiol in the retrochiasmatic area by comparing the effects of estradiol and genistein, a selective ERβ agonist. We also examined whether there are seasonal changes in response of the retrochiasmatic area to these agonists and if these effects are mediated by dopamine. To test these hypotheses, ovariectomized ewes were implanted with bilateral guide cannulae targeting the retrochiasmatic area. Crystalline agonists were...

Thyroid hormones permit the increase in response to estradiol negative feedback in ewes at the transition to anestrus. In this study, we tested whether the thyroid hormones are also required for steroid-independent seasonal changes in pulsatile LH secretion. In experiment 1, Suffolk ewes were ovariectomized and thyroidectomized (THX) or ovariectomized only (controls) in late November. LH pulse frequency and amplitude were measured for 4 h in December, April, May, June, and August. Pulse frequency was also measured in the presence of estradiol-containing implants during the breeding (December) and early anestrus (March) seasons. As expected, in the presence of estradiol, pulse frequency declined between December and March in control but not THX ewes. In the absence of estradiol, a seasonal decline in frequency and an increase in amplitude occurred in control ewes, concurrent with lengthening photoperiod. A similar trend was seen in THX ewes, but the seasonal changes were lower in magnitude and not significant. In experiment 2, the same protocol was used (pulse measurements in December, May, and June) with a larger THX group size (n = 7). Results were similar to those of experiment 1 for controls. In THX ewes, pulse frequency did not change over time and was significantly elevated relative to that of controls during the summer. Pulse amplitude in THX ewes tended to increase during summer and did not differ from pulse amplitudes in control ewes. These results demonstrate that thyroid hormones are required for steroid-independent cycles in LH pulse frequency; however, some seasonal changes in amplitude still occur in the absence of thyroid hormones. This finding contrasts with the changes in estradiol negative feedback at the transition to anestrus, which are entirely thyroid hormone dependent.

Seasonal reproduction is a common adaptive strategy among mammals that allows for breeding to occur at times of the year when it is most advantageous for the subsequent survival and growth of offspring. A major mechanism responsible for seasonal reproduction is a striking increase in the responsiveness of gonadotropin-releasing hormone (GnRH) neurons to the negative feedback effects of estradiol. The neural and neuroendocrine circuitry responsible for mammalian seasonal reproduction has been primarily studied in three animal models: the sheep, and two species of hamsters. In this review, we first describe the afferent signals, neural circuitry and transmitters/peptides responsible for seasonal reproductive transitions in sheep, and then compare these mechanisms with those derived from studies in hamsters. The results suggest common principles as well as differences in the role of specific brain nuclei and neuropeptides, including that of kisspeptin cells of the hypothalamic arcuate nucleus, in regulating seasonal reproduction among mammals.

The discovery of episodic luteinizing hormone (LH) secretion in 1970 initiated considerable effort by reproductive neuroendocrinologists to understand the neural mechanisms underlying the synchronous release of gonadotropin-releasing hormone (GnRH) that drives pulsatile LH secretion. In this chapter, we first review early work that attempted to define the neural elements of this GnRH pulse generator, including evidence that it may be endogenous to GnRH neurons. We next describe the development of a new model for the GnRH pulse generator based on KNDy neurons in the arcuate nucleus that contain kisspeptin, neurokinin B, and dynorphin, and consider recent experimental tests of this model, first in sheep and goats, then in rats and mice. Based on these studies, we suggest a modified model for the GnRH pulse generator in ruminants and rodents, and discuss whether this model is applicable to humans.

Oestradiol exerts a season-specific negative feedback effect on the GnRH/LH neurosecretory system of the Suffolk ewe. This neuroendocrine suppression is mediated in part by dopamine A15 neurones, but these neurones do not possess the oestrogen receptor. Based on indirect evidence, we hypothesized that oestrogen receptor-containing neurones in the ventromedial preoptic area (vmPOA) may be the initial step in a neuronal system whereby oestradiol suppresses GnRH secretion during the non-breeding season. To test this, three experiments were conducted using ovariectomized ewes receiving either empty or oestradiol-containing bilateral microimplants directed at the vmPOA or s.c. subcutaneous oestradiol-containing implants. In the first experiment, LH pulse frequency was measured on days 0, 1, 7 and 14 of treatment during seasonal anoestrus. In vmPOA oestradiol and s.c. oestradiol groups only, LH pulse frequency was suppressed on days 7 and 14, with maximal suppression evident by day 7. In the second experiment, this protocol was repeated during the breeding season, with LH pulses examined on days 0 and 7; LH pulse frequency did not change in any group. The third experiment tested if the effect of vmPOA oestradiol during anoestrus could be overcome by an injection of the dopamine-D2 receptor antagonist (-)-sulpiride. The vmPOA microimplants and s.c. oestradiol implants again suppressed LH pulse frequency and this was reversed by sulpiride in vmPOA oestradiol ewes. We conclude that oestradiol acts on cells in the vmPOA to stimulate a system involving dopamine neurones that inhibits GnRH/LH pulsatility in the anoestrous ewe.