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.

Evidence that orphanin FQ mediates progesterone negative feedback in the ewe.

Orphanin FQ (OFQ), a member of the opioid family, is found in many areas of the hypothalamus and, when given centrally OFQ inhibits episodic LH secretion in rodents and sheep. Because GnRH neurons are devoid of the appropriate receptors to mediate steroid negative feedback directly, neurons that release OFQ may be involved. Using immunocytochemistry, we first determined that most OFQ neurons in the arcuate nucleus (ARC) and other hypothalamic regions of luteal phase ewes contained both estrogen receptor α and progesterone (P) receptor. Given a similar high degree of steroid receptor colocalization in other ARC subpopulations, we examined whether OFQ neurons of the ARC contained those other neuropeptides and neurotransmitters. OFQ did not colocalize with kisspeptin, tyrosine hydroxylase, or agouti-related peptide, but all ARC OFQ neurons coexpressed proopiomelanocortin. To test for a role for endogenous OFQ, we examined the effects of an OFQ receptor antagonist, [Nphe1,Arg14,Lys15]Nociceptin-NH2 (UFP-101) (30 nmol intracerebroventricular/h), on LH secretion in steroid-treated ewes in the breeding season and ovary-intact ewes in anestrus. Ovariectomized ewes with luteal phase concentrations of P and estradiol showed a significant increase in LH pulse frequency during infusion of UFP-101 (4.5 ± 0.5 pulses/6 h) compared with saline infusion (2.6 ± 0.4 pulses/6 h), whereas ewes implanted with only estradiol did not. Ovary-intact anestrous ewes displayed no significant differences in LH pulse amplitude or frequency during infusion of UFP-101. Therefore, we conclude that OFQ mediates, at least in part, the negative feedback action of P on GnRH/LH pulse frequency in sheep.

Kisspeptin, neurokinin B, and dynorphin act in the arcuate nucleus to control activity of the GnRH pulse generator in ewes.

Recent work has led to the hypothesis that kisspeptin/neurokinin B/dynorphin (KNDy) neurons in the arcuate nucleus play a key role in GnRH pulse generation, with kisspeptin driving GnRH release and neurokinin B (NKB) and dynorphin acting as start and stop signals, respectively. In this study, we tested this hypothesis by determining the actions, if any, of four neurotransmitters found in KNDy neurons (kisspeptin, NKB, dynorphin, and glutamate) on episodic LH secretion using local administration of agonists and antagonists to receptors for these transmitters in ovariectomized ewes. We also obtained evidence that GnRH-containing afferents contact KNDy neurons, so we tested the role of two components of these afferents: GnRH and orphanin-FQ. Microimplants of a Kiss1r antagonist briefly inhibited LH pulses and microinjections of 2 nmol of this antagonist produced a modest transitory decrease in LH pulse frequency. An antagonist to the NKB receptor also decreased LH pulse frequency, whereas NKB and an antagonist to the receptor for dynorphin both increased pulse frequency. In contrast, antagonists to GnRH receptors, orphanin-FQ receptors, and the N-methyl-D-aspartate glutamate receptor had no effect on episodic LH secretion. We thus conclude that the KNDy neuropeptides act in the arcuate nucleus to control episodic GnRH secretion in the ewe, but afferent input from GnRH neurons to this area does not. These data support the proposed roles for NKB and dynorphin within the KNDy neural network and raise the possibility that kisspeptin contributes to the control of GnRH pulse frequency in addition to its established role as an output signal from KNDy neurons that drives GnRH pulses.

Evidence that the arcuate nucleus is an important site of progesterone negative feedback in the ewe.

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.

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.

Neurokinin B acts via the neurokinin-3 receptor in the retrochiasmatic area to stimulate luteinizing hormone secretion in sheep.

Recent data have demonstrated that mutations in the receptor for neurokinin B (NKB), the NK-3 receptor (NK3R), produce hypogonadotropic hypogonadism in humans. These data, together with reports that NKB expression increases after ovariectomy and in postmenopausal women, have led to the hypothesis that this tachykinin is an important stimulator of GnRH secretion. However, the NK3R agonist, senktide, inhibited LH secretion in rats and mice. In this study, we report that senktide stimulates LH secretion in ewes. A dramatic increase in LH concentrations to levels close to those observed during the preovulatory LH surge was observed after injection of 1 nmol senktide into the third ventricle during the follicular, but not in the luteal, phase. Similar increases in LH secretion occurred after insertion of microimplants containing this agonist into the retrochiasmatic area (RCh) in anestrous or follicular phase ewes. A low-dose microinjection (3 pmol) of senktide into the RCh produced a smaller but significant increase in LH concentrations in anestrous ewes. Moreover, NK3R immunoreactivity was clearly evident in the RCh, although it was not found in A15 dopaminergic cell bodies in this region. These data provide evidence that NKB stimulates LH (and presumably GnRH) secretion in ewes and point to the RCh as one important site of action. Based on these data, and the effects of NK3R mutations in humans, we hypothesize that NKB plays an important stimulatory role in the control of GnRH and LH secretion in nonrodent species.

Estradiol negative feedback regulation by glutamatergic afferents to A15 dopaminergic neurons: variation with season.

It is now clear that seasonal breeding in ewes is due to an increase in response to estradiol (E(2)) negative feedback in the nonbreeding season (anestrus) that is mediated by the A15 group of dopaminergic (DA) neurons. Because A15 cells do not contain estrogen receptors, we have postulated the presence of estrogen-responsive afferents and recently reported evidence that input from neurons containing gamma-aminobutyric acid (GABA) contribute to the control of A15 activity by E(2). However, GABAergic afferents account for only a fraction of A15 synaptic input and do not appear to vary with season. We therefore investigated the possible role of stimulatory glutamatergic input to A15 neurons. In experiments 1 and 2, local administration into the A15 of either a N-methyl-D-aspartate (NMDA) receptor or a kainate/alpha-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA) receptor antagonist stimulated episodic LH secretion in a dose-dependent manner in ovary-intact anestrous ewes. In experiment 3, we examined the number of glutamatergic close contacts onto A15 neurons using dual immunocytochemistry in tissue from E(2)-treated ovariectomized anestrous and breeding season ewes. All A15 DA neurons were contacted by glutamatergic vesicles, and the number of close contacts was significantly higher in anestrus than the breeding season. Finally, using a triple-label immunocytochemistry procedure, we did not observe any colocalization of markers for GABA and glutamate in vesicles contacting A15 neurons. These results support the hypothesis that glutamatergic afferents actively stimulate A15 DA neurons in ovary-intact anestrous ewes and raise the possibility that alterations in this input may contribute to increased A15 neural activity during anestrus.

Evidence that gamma-aminobutyric acid is part of the neural circuit mediating estradiol negative feedback in anestrous ewes.

Seasonal anestrus in ewes is driven by an increase in response to estradiol (E2) negative feedback. Compelling evidence indicates that inhibitory A15 dopaminergic (DA) neurons mediate the increased inhibitory actions of E2 in anestrus, but these neurons do not contain estrogen receptors. Therefore, we have proposed that estrogen-responsive afferents to A15 neurons are part of the neural circuit mediating E2 negative feedback in anestrus. This study examined the possible role of afferents containing gamma-aminobutyric acid (GABA) and nitric oxide (NO) in modulating the activity of A15 neurons. Local administration of NO synthase inhibitors to the A15 had no effect on LH, but GABA receptor ligands produced dramatic changes. Administration of either a GABA A or GABA B receptor agonist to the A15 increased LH secretion in ovary-intact ewes, suggesting that GABA inhibits A15 neural activity. In ovariectomized anestrous ewes, the same doses of GABA receptor agonist had no effect, but combined administration of a GABA A and GABA B receptor antagonist to the A15 inhibited LH secretion. These data are consistent with the hypothesis that endogenous GABA release within the A15 is low in ovary-intact anestrous ewes and elevated after ovariectomy. Using dual immunocytochemistry, we observed that GABAergic varicosities make close contacts on to A15 neurons and that A15 neurons contain both the GABA A-alpha1 and the GABA B-R1 receptor subunits. Based on these data, we propose that in anestrous ewes, E2 inhibits release of GABA from afferents to A15 DA neurons, increasing the activity of these DA neurons and thus suppressing episodic secretion of GnRH and LH.

Inhibition of luteinizing hormone secretion by localized administration of estrogen, but not dihydrotestosterone, is enhanced in the ventromedial hypothalamus during feed restriction in the young wether.

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.

Evidence that dynorphin plays a major role in mediating progesterone negative feedback on gonadotropin-releasing hormone neurons in sheep.

Endogenous opioid peptides (EOP) mediate progesterone-negative feedback in many species, but the specific EOP systems involved remain unresolved. We first addressed this question in sheep by determining the role of different EOP receptor subtypes in the medial basal hypothalamus (MBH) and preoptic area (POA). Local administration of EOP receptor antagonists to luteal phase ewes indicated that kappa-, but not micro- or delta-, receptors mediate the inhibition of LH secretion in the MBH. In contrast, both kappa- and micro-, but not delta-receptor, antagonists increased LH pulse frequency when placed in the POA. We next examined close appositions between dynorphin (kappa ligand) and beta-endorphin (micro ligand) containing varicosities and GnRH perikarya in luteal phase ewes using dual immunocytochemistry and light microscopy. Approximately 90% of MBH GnRH neurons had close associations by dynorphin-containing varicosities, but only 40-50% of GnRH perikarya elsewhere had such close associations. In contrast, the percentage of beta-endorphinergic varicosities close to GnRH neurons was similar among all regions. Electron microscopic analysis demonstrated both dynorphinergic synapses and beta-endorphinergic synapses onto GnRH perikarya. These and other data lead to the hypothesis that dynorphin neurons play a major role in progesterone-negative feedback in the ewe and that this inhibition may be exerted directly on GnRH perikarya within the MBH, whereas dynorphin and beta-endorphin input to GnRH neurons in the POA provide redundancy to this system or are involved in other actions of progesterone or estradiol in the control of the GnRH surge.

Evidence that thyroid hormones act in the ventromedial preoptic area and the premammillary region of the brain to allow the termination of the breeding season in the ewe.

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.

Evidence that estrogen receptor alpha, but not beta, mediates seasonal changes in the response of the ovine retrochiasmatic area to estradiol.

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 (ERalpha). Therefore, we tested the hypothesis that beta-estrogen receptors mediate the action of estradiol in the retrochiasmatic area by comparing the effects of estradiol and genistein, a selective ERbeta 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 administered via microimplants inserted down the cannulae. Blood samples taken before and 4 days after microimplant insertion were analyzed for LH concentrations, pulse frequency, and amplitude. Genistein treatment produced no significant change in LH levels in either season. Estradiol treatment decreased both mean LH concentrations and pulse frequency in anestrous but not breeding-season ewes. Administration of the dopamine antagonist sulpiride to ovariectomized ewes with estradiol microimplants in the retrochiasmatic area returned LH pulse frequency to levels indistinguishable from controls. From these data, we hypothesize that estradiol acts on local ERalpha-containing neurons in this area to stimulate a dopaminergic pathway that inhibits LH secretion during anestrus.

Temporal requirements of thyroid hormones for seasonal changes in LH secretion.

The transition between breeding and anestrous seasons in ewes is driven by an endogenous rhythm in responsiveness to estradiol negative feedback. One stage of this rhythm, the transition to anestrus, requires the presence of thyroid hormone during a window of responsiveness that opens in the late breeding season. The primary goal of this study was to assess when ewes lose responsiveness to thyroid hormone (i.e. when the window closes). In addition, we investigated whether thyroid hormone influences aspects of seasonality other than the transition to anestrus. Ovariectomized ewes maintained in a simulated natural photoperiod were implanted with estradiol, thyroidectomized, and treated with T(4) for 100 d beginning at progressively later dates during the anestrous season. Onset of neuroendocrine anestrus (decrease in LH), latency to anestrus, and time of onset of the subsequent neuroendocrine breeding season (rise in LH) were determined. Ewes gradually lost responsiveness to T(4) during the latter half of the anestrous season, as judged by increasing latency to the decrease in LH and, eventually, failure to exhibit a decrease in LH. Progressively later T(4) replacements also caused progressive delays in the subsequent breeding season. In contrast, the annual PRL cycle was not significantly affected by thyroidectomy or T(4) replacement. These findings indicate that 1) responsiveness to T(4) is lost gradually during the mid to late anestrous season; 2) thyroid hormones can influence the timing of the breeding season and thus may be required for the maintenance or entrainment of the endogenous reproductive rhythm; 3) thyroid hormones are not required for all seasonal neuroendocrine cycles.

Thyroid hormones mediate steroid-independent seasonal changes in luteinizing hormone pulsatility in the ewe.

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.