The effect of NK3-Saporin injection within the arcuate nucleus on puberty, the LH surge, and the response to Senktide in female sheep†.

The timing of puberty onset is reliant on increased gonadotropin-releasing hormone (GnRH). This elicits a corresponding increase in luteinizing hormone (LH) due to a lessening of sensitivity to the inhibitory actions of estradiol (E2). The mechanisms underlying the increase in GnRH release likely involve a subset of neurons within the arcuate (ARC) nucleus of the hypothalamus that contain kisspeptin, neurokinin B (NKB), and dynorphin (KNDy neurons). We aimed to determine if KNDy neurons in female sheep are critical for: timely puberty onset; the LH surge; and the response to an intravenous injection of the neurokinin-3 receptor (NK3R) agonist, senktide. Prepubertal ewes received injections aimed at the ARC containing blank-saporin (control, n = 5) or NK3-saporin (NK3-SAP, n = 6) to ablate neurons expressing NK3R. Blood samples taken 3/week for 65 days following surgery were assessed for progesterone to determine onset of puberty. Control ewes exhibited onset of puberty at 33.2 ± 3.9 days post sampling initiation, whereas 5/6 NK3-SAP treated ewes didn't display an increase in progesterone. After an artificial LH surge protocol, surge amplitude was lower in NK3-SAP ewes. Finally, ewes were treated with senktide to determine if an LH response was elicited. LH pulses were evident in both groups in the absence of injections, but the response to senktide vs saline was similar between groups. These results show that KNDy cells are necessary for timely puberty onset and for full expresson of the LH surge. The occurrence of LH pulses in NK3-SAP treated ewes may indicate a recovery from an apulsatile state.

Regulation of GnRH pulsatility in ewes.

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 E2 decreasing kisspeptin and progesterone increasing dynorphin release onto GnRH neurons. In pre-pubertal lambs, E2 inhibits 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; E2 increases dopamine release from these neurons to inhibit KNDy neurons and suppress the frequency of kisspeptin and GnRH release.

Neuroanatomical Relationship of Neuronal Nitric Oxide Synthase to Gonadotropin-Releasing Hormone and Kisspeptin Neurons in Adult Female Sheep and Primates.

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.

Neural mechanisms controlling seasonal reproduction: principles derived from the sheep model and its comparison with hamsters.

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.

A role for neurokinin B in pulsatile GnRH secretion in the ewe.

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.

Neuroanatomy of the kisspeptin signaling system in mammals: comparative and developmental aspects.

Our understanding of kisspeptin and its actions depends, in part, on a detailed knowledge of the neuroanatomy of the kisspeptin signaling system in the brain. In this chapter, we will review our current knowledge of the distribution of kisspeptin cells, fibers, and receptors in the mammalian brain, including the development, phenotype, and projections of different kisspeptin subpopulations. A fairly consistent picture emerges from this analysis. There are two major groups of kisspeptin cell bodies: a large number in the arcuate nucleus (ARC) and a smaller collection in the rostral periventricular area of the third ventricle (RP3V) of rodents and preoptic area (POA) of non-rodents. Both sets of neurons project to GnRH cell bodies, which contain Kiss1r, and the ARC kisspeptin population also projects to GnRH axons in the median eminence. ARC kisspeptin neurons contain neurokinin B and dynorphin, while a variable percentage of those cells in the RP3V of rodents contain galanin and/or dopamine. Neurokinin B and dynorphin have been postulated to contribute to the control of GnRH pulses and sex steroid negative feedback, while the role of galanin and dopamine in rostral kisspeptin neurons is not entirely clear. Kisspeptin neurons, fibers, and Kiss1r are found in other areas, including widespread areas outside the hypothalamus, but their physiological role(s) in these regions remains to be determined.

KNDy neurons as the GnRH pulse generator: Recent studies in ruminants.

This review considers three aspects of recent work on the role of KNDy neurons in GnRH pulse generation in ruminants. First, work on basic mechanisms of pulse generation includes several tests of this hypothesis, all of which support it, and evidence that Kiss1r-containing neurons form a positive feedback circuit with the KNDy neural network that strengthen the activity of this network. The second section on pathways mediating external inputs focuses on the influence of nutrition and photoperiod, and describes the evidence supporting roles for proopiomelanocortin (POMC) and agouti-related peptide (AgRP) afferents to KNDy cells in each of these. Finally, we review studies exploring the potential applications of manipulating signaling by kisspeptin, and the other KNDy peptides, to control reproductive function in domestic animals and conclude that, although these approaches show some promise, they do not have major advantages over current practices at this time.

Lesions of KNDy and Kiss1R Neurons in the Arcuate Nucleus Produce Different Effects on LH Pulse Patterns in Female Sheep.

The current model for the synchronization of GnRH neural activity driving GnRH and LH pulses proposes that a set of arcuate (ARC) neurons that contain kisspeptin, neurokinin B, and dynorphin (KNDy neurons) is the GnRH pulse generator. This study tested the functional role of ovine KNDy neurons in pulse generation and explored the roles of nearby Kiss1 receptor (Kiss1R)-containing cells using lesions produced with saporin (SAP) conjugates. Injection of NK3-SAP ablated over 90% of the KNDy cells, while Kiss-SAP (saporin conjugated to kisspeptin-54) lesioned about two-thirds of the Kiss1R population without affecting KNDy or GnRH cell number. Both lesions produced a dramatic decrease in LH pulse amplitude but had different effects on LH pulse patterns. NK3-SAP increased interpulse interval, but Kiss-SAP did not. In contrast, Kiss-SAP disrupted the regular hourly occurrence of LH pulses, but NK3-SAP did not. Because Kiss1R is not expressed in KNDy cells, HiPlex RNAScope was used to assess the colocalization of 8 neurotransmitters and 3 receptors in ARC Kiss1R-containing cells. Kiss1R cells primarily contained transcript markers for GABA (68%), glutamate (28%), ESR1 (estrogen receptor-α) mRNA, and OPRK1 (kappa opioid receptor) mRNA. These data support the conclusion that KNDy neurons are essential for GnRH pulses in ewes, whereas ARC Kiss1R cells are not but do maintain the amplitude and regularity of GnRH pulses. We thus propose that in sheep, ARC Kiss1R neurons form part of a positive feedback circuit that reinforces the activity of the KNDy neural network, with GABA or glutamate likely being involved.

Neuroendocrine control of gonadotropin-releasing hormone: Pulsatile and surge modes of secretion.

The concept that different systems control episodic and surge secretion of gonadotropin-releasing hormone (GnRH) was well established by the time that GnRH was identified and formed the framework for studies of the physiological roles of GnRH, and later kisspeptin. Here, we focus on recent studies identifying the neural mechanisms underlying these two modes of secretion, with an emphasis on their core components. There is now compelling data that kisspeptin neurons in the arcuate nucleus that also contain neurokinin B (NKB) and dynorphin (i.e., KNDy cells) and their projections to GnRH dendrons constitute the GnRH pulse generator in mice and rats. There is also strong evidence for a similar role for KNDy neurons in sheep and goats, and weaker data in monkeys and humans. However, whether KNDy neurons act on GnRH dendrons and/or GnRH soma and dendrites that are found in the mediobasal hypothalamus (MBH) of these species remains unclear. The core components of the GnRH/luteinising hormone surge consist of an endocrine signal that initiates the process and a neural trigger that drives GnRH secretion during the surge. In all spontaneous ovulators, the core endocrine signal is a rise in estradiol secretion from the maturing follicle(s), with the site of estrogen positive feedback being the rostral periventricular kisspeptin neurons in rodents and neurons in the MBH of sheep and primates. There is considerable species variations in the neural trigger, with three major classes. First, in reflex ovulators, this trigger is initiated by coitus and carried to the hypothalamus by neural or vascular pathways. Second, in rodents, there is a time of day signal that originates in the suprachiasmatic nucleus and activates rostral periventricular kisspeptin neurons and GnRH soma and dendrites. Finally, in sheep nitric oxide-producing neurons in the ventromedial nucleus, KNDy neurons and rostral kisspeptin neurons all appear to participate in driving GnRH release during the surge.

A 10-year follow-up of treatment outcomes in patients with early stage breast cancer and clinically negative axillary nodes treated with tangential breast irradiation following sentinel lymph node dissection or axillary clearance.

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.

Neurons of the lateral preoptic area/rostral anterior hypothalamic area are required for photoperiodic inhibition of estrous cyclicity in sheep.

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.

Evidence that synaptic plasticity of glutamatergic inputs onto KNDy neurones during the ovine follicular phase is dependent on increasing levels of oestradiol.

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.

Neuronal plasticity and seasonal reproduction in sheep.

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.

Morphological and functional evidence for sexual dimorphism in neurokinin B signalling in the retrochiasmatic area of 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.

Evidence that Nitric Oxide Is Critical for LH Surge Generation 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.

Kisspeptin and seasonality in sheep.

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.

Does the KNDy Model for the Control of Gonadotropin-Releasing Hormone Pulses Apply to Monkeys and Humans?

There is now considerable evidence supporting the role of a subpopulation of neurons in the arcuate nucleus of the hypothalamus that coexpress kisspeptin, neurokinin B, and dynorphin (abbreviated as KNDy neurons) as the long sought-after gonadotropin-releasing hormone (GnRH) pulse generator. The "KNDy hypothesis" of pulse generation has largely been based on findings in rodents and ruminants, and there is considerably less information about the anatomical and functional organization of the KNDy subpopulation in the primate hypothalamus. In this review, we focus on the applicability of this hypothesis, and the roles of kisspeptin, neurokinin B, and dynorphin in reproduction, to humans and nonhuman primates, reviewing available data and pointing out important gaps in our current knowledge. With recent application of drugs that target KNDy peptides and their receptors to therapeutic treatments for reproductive disorders, it is imperative we fully understand the primate KNDy network and its role in the control of GnRH secretion, as well as species differences in this system that may exist between humans, nonhuman primates, and other mammals.

Prenatal Testosterone Exposure Alters GABAergic Synaptic Inputs to GnRH and KNDy Neurons in a Sheep Model of Polycystic Ovarian Syndrome.

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.

Evidence That the LH Surge in Ewes Involves Both Neurokinin B-Dependent and -Independent Actions of Kisspeptin.

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.