Preoptic Area Kisspeptin Research Articles

Estrogen increases KISS1 expression in newly generated immortalized KISS1-expressing cell line derived from goat preoptic area.

Kisspeptin neurons located in the hypothalamic preoptic area (POA) are suggested to be responsible for the induction of the gonadotropin-releasing hormone (GnRH) surge and the following luteinizing hormone (LH) surge to regulate female mammals’ ovulation. Accumulating evidence demonstrates that the preovulatory level of estrogen activates the POA kisspeptin neurons (estrogen positive feedback), which in turn induces a GnRH/LH surge. This study aimed to derive a cell line from goat POA kisspeptin neurons as an in vitro model to analyze the estrogen positive feedback mechanism in ruminants. Neuron-derived cell clones obtained by the immortalization of POA tissue from a female Shiba goat fetus were analyzed for the expression of kisspeptin (KISS1) and estrogen receptor α (ESR1) genes using quantitative real-time reverse transcription-polymerase chain reaction and three cell clones were selected as POA kisspeptin neuron cell line candidates. One cell line (GP64) out of the three clones showed significant increase in the KISS1 level by incubation with estradiol for 24 h, indicating that the GP64 cells mimic endogenous goat POA kisspeptin neurons. The GP64 cells showed immunoreactivities for kisspeptin and estrogen receptor α and retained a stable growth rate throughout three passages. Further, intracellular calcium levels in the GP64 cells were increased by the KCl challenge, indicating their neurosecretory ability. In conclusion, we generated a new KISS1-expressing cell line derived from goat POA. The current GP64 cell line could be a useful model to elucidate the estrogen positive feedback mechanism responsible for the GnRH/LH surge generation in ruminants.

Expression of progesterone receptor, estrogen receptors α and β, and kisspeptin in the hypothalamus during perinatal development of gonad-lacking steroidogenic factor-1 knockout mice

Gonadal hormones contribute to brain sexual differentiation. We analyzed expression of progesterone receptor (PR), estrogen receptor-α (ERα), ERβ, and kisspeptin, in the preoptic area (POA) and/or the arcuate nucleus (ARC), in gonad-lacking steroidogenic factor-1 knockout (KO) mice during perinatal development. At postnatal-day (P) 0-P7, POA PR levels were higher in wild-type (WT) males compared with WT females, while those in KO males were lower than in WT males and similar to those in WT and KO females. At P14–P21, PR levels in all groups increased similarly. POA ERα levels were similar in all groups at embryonic-day (E) 15.5-P14. Those in WT but not KO males reduced during postnatal development to be significantly lower compared with females at P21. POA ERβ levels were higher in WT males than in WT females, while those in KO males were lower than in WT males and similar to those in WT and KO females at P0–P21. POA kisspeptin expression was female-biased in WT mice, while levels in KO females were lower compared with WT females and similar to those in WT and KO males. ARC kisspeptin levels were equivalent among groups at E15.5-P0. At P7–P21, ARC levels in WT but not KO males became lower compared with WT females. Diethylstilbestrol exposure during P0–P6 and P7–P13 increased POA PR and ERβ, and decreased POA ERα and ARC kisspeptin levels at P7 and/or P14 in both sexes of KO mice. These data further understanding of gonadal hormone action on neuronal marker expression during brain sexual development.

Prenatal testosterone exposure decreases colocalization of insulin receptors in kisspeptin/neurokinin B/dynorphin and agouti-related peptide neurons of the adult ewe.

Insulin serves as a link between the metabolic and reproductive systems, communicating energy availability to the hypothalamus and enabling reproductive mechanisms. Adult Suffolk ewes prenatally exposed to testosterone (T) display an array of reproductive and metabolic dysfunctions similar to those seen in women with polycystic ovarian syndrome (PCOS), including insulin resistance. Moreover, prenatal T treatment alters neuropeptide expression in KNDy (co-expressing kisspeptin, neurokinin B/dynorphin) and agouti-related peptide (AgRP) neurons in the arcuate nucleus, two populations that play key roles in the control of reproduction and metabolism, respectively. In this study, we determined whether prenatal T treatment also altered insulin receptors in KNDy and AgRP neurons, as well as in preoptic area (POA) kisspeptin, pro-opiomelanocortin (POMC), and gonadotropin-releasing hormone (GnRH) neurons of the adult sheep brain. Immunofluorescent detection of the beta subunit of insulin receptor (IRβ) revealed that KNDy, AgRP and POMC neurons, but not GnRH or POA kisspeptin neurons, colocalize IRβ in control females. Moreover, prenatal T treatment decreased the percentage of KNDy and AgRP neurons that colocalized IRβ, consistent with reduced insulin sensitivity. Administration of the anti-androgen drug, Flutamide, during prenatal T treatment, prevented the reduction in IRβ colocalization in AgRP, but not in KNDy neurons, suggesting that these effects are programmed by androgenic and oestrogenic actions, respectively. These findings provide novel insight into the effects of prenatal T treatment on hypothalamic insulin sensitivity and raise the possibility that decreased insulin receptors, specifically within KNDy and AgRP neurons, may contribute to the PCOS-like phenotype of this animal model.

Progesterone-induced amplification and advancement of GnRH/LH surges are associated with changes in kisspeptin system in preoptic area of estradiol-primed female rats

The time course effects of ovarian steroids on kisspeptin and GnRH/LH systems is not totally clarified. We investigated the temporal relationship among kisspeptin and GnRH mRNA and kisspeptin content in the preoptic area (POA), GnRH content and release in the medial basal hypothalamus (MBH) and plasma LH levels under different steroid treatments. Ovariectomized rats treated with oil (OVOO), oil plus single dose of estradiol (OVOE), oil plus single dose of progesterone (OVOP), estradiol for 3 days plus oil (OVEO) or estradiol for 3 days plus progesterone (OVEP) were hourly decapitated from 10:00 to 17:00 or had the MBH microdialyzed from 09:00 to 19:00. Estradiol and progesterone acutely increased POA kisspeptin content without altering POA kisspeptin mRNA levels. Short-term exposure to both hormones stimulated MBH GnRH content, although no GnRH/LH surges had occurred. Chronic estradiol-treatment increased both kisspeptin mRNA levels and content in the POA, demonstrating that long exposure to estradiol is required to activate the whole kisspeptin synthesis machinery. This was followed by the peak in the GnRH/LH release. In estradiol-primed rats, progesterone further increased POA kisspeptin content, amplified and advanced GnRH/LH surges, with no additional change on POA kisspeptin mRNA. The data show an estradiol-induced temporal association between kisspeptin increase in the POA and GnRH/LH surges. Interestingly, the classic action of progesterone in amplifying and accelerating the GnRH/LH surges seems to occur by a mechanism which involves POA kisspeptin system.

Prenatal Testosterone Treatment Leads to Changes in the Morphology of KNDy Neurons, Their Inputs, and Projections to GnRH Cells in Female Sheep.

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.

Evidence for Changes in Numbers of Synaptic Inputs onto KNDy and GnRH Neurones during the Preovulatory LH Surge in the Ewe.

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.

Oestrogen-induced activation of preoptic kisspeptin neurones may be involved in the luteinising hormone surge in male and female Japanese monkeys.

The oestrogen-induced luteinising hormone (LH) surge is evident in male primates, including humans, whereas male rodents never show the LH surge, even when treated with a preovulatory level of oestrogen. This suggests that the central mechanism governing reproductive hormones in primates is different from that in rodents. The present study aimed to investigate whether male Japanese monkeys conserve a brain mechanism mediating the oestrogen-induced LH surge via activation of kisspeptin neurones. Adult male and female Japanese monkeys were gonadectomised and then were treated with oestradiol-17β for 2 weeks followed by a bolus injection of oestradiol benzoate. Both male and female monkeys showed an oestrogen-induced LH surge. In gonadectomised monkeys sacrificed just before the anticipated time of the LH surge, oestrogen treatment significantly increased the number of KISS1-expressing cells in the preoptic area (POA) and enhanced the expression of c-fos in POA KISS1-positive cells of males and females. The oestrogen treatment failed to induce c-fos expression in the arcuate nucleus (ARC) kisspeptin neurones in both sexes just prior to LH surge onset. Thus, kisspeptin neurones in the POA but not in the ARC might be involved in the positive-feedback action of oestrogen that induces LH surge in male Japanese monkeys, as well as female monkeys. The present results indicate that oestrogen-induced activation of POA kisspeptin neurones may contribute to the LH surge generation in both sexes. The conservation of the LH surge generating system found in adult male primates, unlike rodents, could be a result of the capability of oestrogen to induce POA kisspeptin expression and activation.

KNDy (Kisspeptin/Neurokinin B/Dynorphin) Neurons Are Activated during Both Pulsatile and Surge Secretion of LH in the Ewe

KNDy (kisspeptin/neurokinin B/dynorphin) neurons of the arcuate nucleus (ARC) appear to mediate the negative feedback actions of estradiol and are thought to be key regulators of pulsatile LH secretion. In the ewe, KNDy neurons may also be involved with the positive feedback actions of estradiol (E(2)) to induce the LH surge, but the role of kisspeptin neurons in the preoptic area (POA) remains unclear. The goal of this study was to identify which population(s) of kisspeptin neurons is (are) activated during the LH surge and in response to the removal of E(2)-negative feedback, using Fos as an index of neuronal activation. Dual-label immunocytochemistry for kisspeptin and Fos was performed on sections containing the ARC and POA from ewes during the luteal phase of the estrous cycle, or before or after the onset of the LH surge (experiment 1), and from ovary-intact, short-term (24 h) and long-term (>30 d) ovariectomized (OVX) ewes in anestrus (experiment 2). The percentage of kisspeptin neurons expressing Fos in both the ARC and POA was significantly higher during the LH surge. In contrast, the percentage of kisspeptin/Fos colocalization was significantly increased in the ARC, but not POA, after both short- and long-term E(2) withdrawal. Thus, POA kisspeptin neurons in the sheep are activated during, and appear to contribute to, E(2)-positive feedback, whereas ARC kisspeptin (KNDy) neurons are activated during both surge and pulsatile modes of secretion and likely play a role in mediating both positive and negative feedback actions of E(2) on GnRH secretion in the ewe.

Kisspeptin neurons mediate reflex ovulation in the musk shrew ( Suncus murinus )

The present study investigated whether kisspeptin-G protein-coupled receptor 54 (GPR54) signaling plays a role in mediating mating-induced ovulation in the musk shrew (Suncus murinus), a reflex ovulator. For this purpose, we cloned suncus Kiss1 and Gpr54 cDNA from the hypothalamus and found that suncus kisspeptin (sKp) consists of 29 amino acid residues (sKp-29). Injection of exogenous sKp-29 mimicked the mating stimulus to induce follicular maturation and ovulation. Administration of several kisspeptins and GPR54 agonists also induced presumed ovulation in a dose-dependent manner, and Gpr54 mRNA was distributed in the hypothalamus, showing that kisspeptins induce ovulation through binding to GPR54. The sKp-29-induced ovulation was blocked completely by pretreatment with a gonadotropin-releasing hormone (GnRH) antagonist, suggesting that kisspeptin activates GnRH neurons to induce ovulation in the musk shrew. In addition, in situ hybridization revealed that Kiss1-expressing cells are located in the medial preoptic area (POA) and arcuate nucleus in the musk shrew hypothalamus. The number of Kiss1-expressing cells in the POA or arcuate nucleus was up-regulated or down-regulated by estradiol, suggesting that kisspeptin neurons in these regions were the targets of the estrogen feedback action. Finally, mating stimulus largely induced c-Fos expression in Kiss1-positive cells in the POA, indicating that the mating stimulus activates POA kisspeptin neurons to induce ovulation. Taken together, these results indicate that kisspeptin-GPR54 signaling plays a role in the induction of ovulation in the musk shrew, a reflex ovulator, as it does in spontaneous ovulators.

Arcuate Kisspeptin Neurons Play a Key Role in the Feedback Control of GnRH Secretion in Sheep.Robert L. Goodman, Ph.D.

In rodents, two populations of kisspeptin neurons, one in the arcuate nucleus (ARC) and the other in the preoptic area (POA) appear to contribute to the feedback control of GnRH. In large mammals including sheep and primates, the majority of kisspeptin neurons are found in the ARC. In sheep, these kisspeptin neurons have several important characteristics. Anatomically, the ARC kisspeptin neurons also contain dynorphin and neurokinin-B, whereas POA kisspeptin neurons do not. Functionally, ARC kisspeptin neurons appear to play important roles in both the anestrous and breeding season. In anestrus, they become more responsive to inhibition by estradiol and thus likely contribute to the annual changes in estradiol negative feedback that drive seasonal reproduction in ewes. During the breeding season, when estradiol suppresses LH pulse amplitude and progesterone inhibits pulse frequency, kisspeptin mRNA expression in the ARC correlates with estradiol inhibition of pulse amplitude. Progesterone has no effect on kisspeptin mRNA expression suggesting that kisspeptin is not critical for progesterone negative feedback. This conclusion is consistent with previous data indicating that progesterone most likely inhibits LH pulse frequency via dynorphin produced in these same ARC neurons. Based on these data, we propose that one subpopulation of ARC neurons mediate the negative feedback actions of estradiol and progesterone, but do so via different neurotransmitters. Thus ARC kisspeptin neurons appear to be key mediators of steroid negative feedback in ewes; we are currently exploring the possibility that they are also involved in the positive feedback action of estradiol.