Does Kisspeptin Signalling Offer a Novel Therapeutic Target for Infertility

Title: Does Kisspeptin signalling offer a new way to treat infertility?

Channa N Jayasena, Waljit S Dhillo, Stephen R Bloom

Department of Investigative Medicine, Faculty of Medicine, ImperialCollegeLondon, HammersmithHospital, London, UK

Corresponding author for proof and reprints:

Prof. S.R. Bloom

Department of Investigative Medicine,

Faculty of Medicine,

6th Floor, CommonwealthBuilding,

ImperialCollegeLondon,

HammersmithHospital,

Du Cane Road,

London W12 ONN.

Tel: +44 208 383 3242

Fax: +44 208 383 3142

Word count: 1758

Conflict of interests: none declared

Financial disclosure

C.N.J. is supported by a Wellcome Trust Research Training Fellowship. W.S.D. is supported by a Department of Health Clinician Scientist Award. This work is funded by an MRC Experimental Medicine project grant. The department is funded by an Integrative Mammalian Biology (IMB) Capacity Building Award and the NIHR Biomedical Research Centre Funding Scheme.

Over the last 5 years, the field of reproductive biology has seen a surge of interest in the group of peptides termed Kisspeptin. These peptides are notably distributed in the placenta, pituitary gland, as well asspecialised hypothalamic neurons[1;2], and have recently emerged as important regulatory factors for control of the mammalian hypothalamo-pituitary-gonadal (HPG) axis[3;4].Therehas been much speculation over the therapeutic potential of Kisspeptin and its receptor, G-protein coupled receptor 54 (GPR54). But couldfuture infertility therapies which target GPR54, ever become reality?

Kisspeptin: an emerging role in reproduction

In 2003, researchers made the ground-breaking observation that inactivating mutations of GPR54in humans and mice lead to pubertal failure together with hypogonadotrophic hypogonadism (low circulating gonadotrophins and sex steroids)[3;4]. Mutant mice lacking GPR54 had normal gonadotrophin releasing hormone(GnRH) neuronal migration, and responded to exogenous GnRH injection[4].Kiss1 mutant mice display a similar albeit less severe phenotype of hypogonadotrophic hypogonadism[5;6]. Furthermore central administration of aGPR54 antagonist suppresses GnRH release in female pubertal monkeys, reduces both mean LH (luteinising hormone) levels and LH pulse amplitude in adult ovariectomised sheep, and abolishes the post-castration rise in LH seen in adult mice, rats and sheep [7]. Therefore GPR54 signalling plays an important role in pubertal development and adult reproductive physiology.

Numerous investigators have demonstrated that central or peripheral injection of Kisspeptin to all mammalian species studied to date (including rats [8-11], mice[12;13], sheep[13-15], hamsters [16], monkeys[17], pigs [18], cows[19] and humans[20;21]), potently stimulates gonadotrophin release. This phenomenon is abolished by co-administration of the GnRH antagonist acyline[9;17]. Most rodent hypothalamic GnRH neurons express GPR54[8;22].Furthermore Kisspeptin induces GnRH release from hypothalamic rat explants[11], and increases electrophysiological activation of green fluorescent protein (GFP)-tagged GnRH neurons within mouse brain slices[22]. Kisspeptin administration to rodents also induces c-fos (a marker of neuronal activation) immunoreactivity in GnRH cell bodies [8]. Taken together, this evidence suggests that Kisspeptinadministration stimulates endogenous hypothalamic GnRH release. Kisspeptin may also act within the pituitary gland.GPR54 is expressed in the pituitary gland[1;23], and some researchers have found that Kisspeptin administration can directly stimulate gonadotrophin release in vitro[24;25]. Hence the primary site of Kisspeptin’s action appears to be the hypothalamic GnRH neuron, although it remains a possibility that an additional (perhaps modulatory) action exists in the pituitary gland.

How does peripherally administered Kisspeptin gain access to the brain in order to stimulate GnRH release? An action at the median eminence of the hypothalamus seems likely; as one of the circumventricular organs, the median eminence has an incomplete blood-brain barrier and is the location where GnRH nerve terminals release GnRH into the hypophyseal portal circulation[26]. Kisspeptin stimulates GnRH from isolated mediobasal hypothalamus explants from wild-type but not GPR54 knockout mice[27]. Since this effect is not abolished by tetrodotoxin (a potent inhibitor action of neuronal potential formation), this suggests a direct action on GnRH nerve terminals within the median eminence [27]. Fos expression in the median eminence also increases following peripheral Kisspeptin administration. Hence a GPR54-mediated action on GnRH nerve terminals within the median eminence, may explain why peripherally administered Kisspeptin effectively stimulates gonadotrophin release.

The neuroanatomy of Kisspeptin neurons suggests a dual function for Kisspeptin signalling within the central nervous system. In rodents, primates and sheep a population of specialised neurons expressingKisspeptin is located in the hypothalamic arcuate nucleus (Arc) (equivalent to the infundibular nucleus in primates)[12;28;29]. These Arc Kisspeptin neurons are negatively regulated by circulating sex steroids. In rodents, levels of Arc Kiss1 expression fall during the evening of proestrous (coincident with the LH surge) or during an oestrogen and progesterone-induced LH surge [30]; however levels of Arc Kiss1 expression rise after gonadectomy, and subsequently fall if sex steroid replacement is given to these animals[31]. A further population of Kisspeptin neurons is located in the anteroventral periventricular (AVPV) nucleus of rodents and sheep[12;29], which is implicated in generation of the LH surge [32]. AVPV neurons are positively regulated by circulating sex steroids, in contrast to neurons of the Arc. In rodents, AVPV Kiss1 expression falls after ovariectomy[31], and rises during the evening of proestrous or during an oestrogen and progesterone-induced LH surge [30]. Thus AVPV Kisspeptin neurons may play a role in generation of the LH surge in female rodents.Although no functional equivalent of the rodent AVPV is found in primates[33], Kisspeptin may also play a role in the human pre-ovulatory surge[21]. Subcutaneous Kisspeptin administration to healthy women stimulates gonadotrophin release 20-fold more potently in the pre-ovulatory phase of the menstrual cycle than in the follicular phase[21]. The basis for this increased sensitivity to Kisspeptin is currently not known.

Clinical studies: where are we now?

Four years ago it was first demonstrated that exogenous Kisspeptin could stimulate gonadotrophin release in humans[20]. Healthy human males were intravenously infused with Kisspeptin-54 in a double-blinded, placebo controlled crossover study[20]. Dose-dependent increases in plasma gonadotrophins were observed. For example, aKisspeptin-54 infusion rate of 4 pmol/kg/min more than doubled 90 minute mean plasma LH, and increased mean plasma FSH by 18% when compared with saline. Subsequently, the first studies of Kisspeptin-54 administration to women were performed[21]. Subcutaneous bolus injections of Kisspeptin-54 at doses between 0.2 and 6.4nmol/kg dose-dependently increased plasma LH and FSH secretion. LH responses to a given dose of kisspeptin wereabout seven times higher than corresponding FSH responses, which is consistent with rodent data from showing the greater effect on LH than FSH[21].

Preliminary results of a study of Kisspeptin administration within a model of human infertility were presented earlier this year[34]. Patients with hypothalamic amenorrhoea (HA) received subcutaneous bolus injections of either Kisspeptin-54 (6.4nmol/kg) or saline. Kisspeptin potently increased mean luteinising hormone (LH) 48-fold and follicle stimulating hormone (FSH) 16-fold when compared with saline injection. Fascinatingly, subjects with HA were over 4-fold more responsive to Kisspeptin injection than previously studied healthy females in the follicular phase of the menstrual cycle[34].

These initial clinical studies demonstrate the efficacy of Kisspeptin in stimulating gonadotrophin release in healthy and infertile human subjects. Hence Kisspeptin can be regarded as a potential novel therapy for human infertility.

How safe is exogenous Kisspeptinlikely to be?

Levels of circulating endogenous Kisspeptin rise to over 9000pmol/l during the third trimester of normal human pregnancy due to placental production, which is in sharp contrast to the barely detectable plasma levels of Kisspeptin (1.3pmol/l) observed in men and non-pregnant women[35]. It has been suggested thatplacental Kisspeptin negatively regulatestrophoblast invasion of uterine tissue; indeed levels of KISS1 expression are significantly lower in invasive forms of gestational trophoblastic disease such as choriocarcinoma, when compared with hydatiform mole or normal placental tissue[36]. Regardless of the function of placental kisspeptin, the fact that women are exposed to markedly raised endogenous levels throughout pregnancy provides some reassurance about its safety. Consistent with this observation, administration of single injections of Kisspeptin to healthy male and female subjects temporarily raised plasma Kisspeptin to levels comparable to those seen in pregnancy,and caused no adverse or behavioural effects such as changes in libido (assessed by questionnaire)[20;21]. No significant changes in heart rate or blood pressure were observed during these studies[20;21], which is noteworthy given that vasoconstrictor properties of Kisspeptin have been demonstrated in vitro[37].

It is important to considerthe specificity of Kisspeptin’s actions. Until recently the only known humoral action of Kisspeptin was on the HPG axis. However evidence from cows [19;38;39]and rats[24] is emerging of a stimulatory action on pituitary growth hormone release. In addition to the pituitary, hypothalamus and placenta, GPR54isexpressed in other regions of the brain as well as the spleen, liver, pancreas and gonads[1;2]. We must therefore remain open to the possibility that additional biological actions of Kisspeptin exist; indeed, effects on insulin secretion [40] and hippocampal synaptic function [41]have been suggested.

What advantages might Kisspeptinhave over existing therapies?

Since clinical studies are at their early stage, we are limited in our ability to judge whether Kisspeptin is likely to provide a novel means of treating human infertility. However the available evidence does at least point to several potential advantages of a therapeutic which targeted GPR54.

As previously discussed, Kisspeptin appears to primarily act by through stimulation of endogenous GnRH release, which in turn triggers endogenous gonadotrophin release[8;9;11;17;22].GPR54 agonistsare therefore predicted to confer a lower risk of excessive ovarian stimulation than gonadotrophin injections[42;43].

No orally bioavailable GPR54 agonist currently exists, but its future discovery might offer a clear practical advantage over GnRH and gonadotrophin-based therapies. A series of pentapeptide-based GPR54 agonists have been described[44;45]. Furthermore small molecule GPR54 agonists have been identified, although detailed results of these have not been published[46].

GPR54 super-agonists may offer a novel means of inducing medical castration. Continuous exposure of monkeys [47]and rodents [48]to exogenous Kisspeptin leads to desensitisation of gonadotrophin responses after 3 hours and 1 day of commencement of administration, respectively. Such desensitisation is probably occurring at the GPR54 receptor since monkeys remained responsive to exogenous GnRH at the end of continuous Kisspeptin infusion[47]. Reduced Kiss1 expression is associated with metastasis in a number of solid tumours[49]. Furthermore KISS1 expression in purified human trophoblasts is significantly lower inchoriocarcinoma when compared withhydatidifom mole or normal placental tissue[36]. It would be fascinating to see if Kisspeptin functions as an anti-metastatic factor in hormone-sensitive tumours; an agent which inhibited tumour metastasis as well as sex-steroid secretion would clearly have therapeutic potential.

It is important to recognise that Kisspeptin would offer no benefit to certain individuals, including those with incompetent GnRH function (such as the defective GnRH neuronal migration observed in Kallman’s syndrome[50]) or a structural pituitary lesion. Furthermore Kisspeptin would be predicted to offer no benefit in post-menopausal women, who already have raised circulating levels of gonadotrophins.

Concluding remarks

We are now beginning to understand the importance of GPR54 signalling. It is obligatory for pubertal development [3;4]and it plays an important role in adult reproductive physiology[7;9;12;13;17]. Initial clinical studies suggest that Kisspeptin potently and safely stimulates gonadotrophin release when administered to healthy volunteers[20;21]. Furthermore a preliminary report has emerged of its efficacy in a human model of infertility[34].The development andfuture availability ofGPR54-based fertility and oncology therapeutics has become very real possibility.Further studies are needed to determine if such agents will offer a distinct advantage over existing therapies.

Reference List

[1] Muir AI, Chamberlain L, Elshourbagy NAet al. AXOR12, a novel human G protein-coupled receptor, activated by the peptide KiSS-1. J.Biol.Chem. 276(31), 28969-28975 (2001).

[2]Ohtaki T, Shintani Y, Honda Set al. Metastasis suppressor gene KiSS-1 encodes peptide ligand of a G-protein-coupled receptor. Nature 411(6837), 613-617 (2001).

[3] de Roux N., Genin E, Carel JC et al. Hypogonadotropic hypogonadism due to loss of function of the KiSS1-derived peptide receptor GPR54. Proc.Natl.Acad.Sci.U.S.A 100(19), 10972-10976 (2003).

** The first evidence that GPR54 signalling plays an important role in human reproduction.

[4] Seminara SB, Messager S, Chatzidaki EE et al. The GPR54 gene as a regulator of puberty. N.Engl.J.Med. 349(17), 1614-1627 (2003).

** Paper providing evidence that GPR54 signalling plays an essential role in pubertal development for both humans and rodents.

[5] Lapatto R, Pallais JC, Zhang D et al. Kiss1-/- mice exhibit more variable hypogonadism than Gpr54-/- mice. Endocrinology 148(10), 4927-4936 (2007).

[6] d'Anglemontde Tassigny X, Fagg LA, Dixon JP et al. Hypogonadotropic hypogonadism in mice lacking a functional Kiss1 gene. Proc.Natl.Acad.Sci.U.S.A 104(25), 10714-10719 (2007).

[7] Roseweir AK, Kauffman AS, Smith JT et al. Discovery of potent kisspeptin antagonists delineate physiological mechanisms of gonadotropin regulation. J.Neurosci. 29(12), 3920-3929 (2009).

** This recent publication describes the first study of a GPR54 antagonist on reprodcutive function in a variety of mammalian species.

[8] Irwig MS, Fraley GS, Smith JT et al. Kisspeptin activation of gonadotropin releasing hormone neurons and regulation of KiSS-1 mRNA in the male rat. Neuroendocrinology 80(4), 264-272 (2004).

[9] Matsui H, Takatsu Y, Kumano S et al. Peripheral administration of metastin induces marked gonadotropin release and ovulation in the rat. Biochem.Biophys.Res.Commun. 320(2), 383-388 (2004).

[10] Navarro VM, Castellano JM, Fernandez-Fernandez R et al. Developmental and hormonally regulated messenger ribonucleic acid expression of KiSS-1 and its putative receptor, GPR54, in rat hypothalamus and potent luteinizing hormone-releasing activity of KiSS-1 peptide. Endocrinology 145(10), 4565-4574 (2004).

** Demonstration that Kisspeptin administration potently stimulates gonadotrophin releasein rodents.

[11] Thompson EL, Patterson M, Murphy KG et al. Central and peripheral administration of kisspeptin-10 stimulates the hypothalamic-pituitary-gonadal axis. J.Neuroendocrinol. 16(10), 850-858 (2004).

[12] Gottsch ML, Cunningham MJ, Smith JT et al. A role for kisspeptins in the regulation of gonadotropin secretion in the mouse. Endocrinology 145(9), 4073-4077 (2004).

[13] Messager S, Chatzidaki EE, Ma D et al. Kisspeptin directly stimulates gonadotropin-releasing hormone release via G protein-coupled receptor 54. Proc.Natl.Acad.Sci.U.S.A 102(5), 1761-1766 (2005).

[14] Caraty A, Smith JT, Lomet D et al. Kisspeptin synchronizes preovulatory surges in cyclical ewes and causes ovulation in seasonally acyclic ewes. Endocrinology 148(11), 5258-5267 (2007).

[15] Arreguin-Arevalo JA, Lents CA, Farmerie TA et al. KiSS-1 peptide induces release of LH by a direct effect on the hypothalamus of ovariectomized ewes. Anim Reprod.Sci. 101(3-4), 265-275 (2007).

[16] Revel FG, Saboureau M, Masson-Pevet M et al. Kisspeptin mediates the photoperiodic control of reproduction in hamsters. Curr.Biol. 16(17), 1730-1735 (2006).

[17] Shahab M, Mastronardi C, Seminara SB et al. Increased hypothalamic GPR54 signaling: a potential mechanism for initiation of puberty in primates. Proc.Natl.Acad.Sci.U.S.A 102(6), 2129-2134 (2005).

[18] Lents CA, Heidorn NL, Barb CR et al. Central and peripheral administration of kisspeptin activates gonadotropin but not somatotropin secretion in prepubertal gilts. Reproduction. 135(6), 879-887 (2008).

[19] Kadokawa H, Matsui M, Hayashi K et al. Peripheral administration of kisspeptin-10 increases plasma concentrations of GH as well as LH in prepubertal Holstein heifers. J.Endocrinol. 196(2), 331-334 (2008).

[20] Dhillo WS, Chaudhri OB, Patterson M et al. Kisspeptin-54 stimulates the hypothalamic-pituitary gonadal axis in human males. J Clin.Endocrinol.Metab (2005).

** The first study of Kisspeptin administration to humans.

[21] Dhillo WS, Chaudhri OB, Thompson EL et al. Kisspeptin-54 stimulates gonadotropin release most potently during the preovulatory phase of the menstrual cycle in women. J Clin.Endocrinol.Metab 92(10), 3958-3966 (2007).

** The first study of Kisspeptin administration to women, revealing differential sensitivity to Kisspeptin across phases of the menstrual cycle.

[22] Han SK, Gottsch ML, Lee KJ et al. Activation of gonadotropin-releasing hormone neurons by kisspeptin as a neuroendocrine switch for the onset of puberty. J Neurosci. 25(49), 11349-11356 (2005).

** Evidence of electrophysiological activation of GnRH neurons by Kisspeptin.

[23] Kotani M, Detheux M, Vandenbogaerde A et al. The metastasis suppressor gene KiSS-1 encodes kisspeptins, the natural ligands of the orphan G protein-coupled receptor GPR54. J.Biol.Chem. 276(37), 34631-34636 (2001).

[24] Gutierrez-Pascual E, Martinez-Fuentes AJ, Pinilla L et al. Direct pituitary effects of kisspeptin: activation of gonadotrophs and somatotrophs and stimulation of luteinising hormone and growth hormone secretion. J.Neuroendocrinol. 19(7), 521-530 (2007).

** An examination of direct humoral effects of Kisspeptin on pituitary cells in vitro.

[25] Suzuki S, Kadokawa H, Hashizume T. Direct kisspeptin-10 stimulation on luteinizing hormone secretion from bovine and porcine anterior pituitary cells. Anim Reprod.Sci. 103(3-4), 360-365 (2008).

[26] Herbison AE. Multimodal influence of estrogen upon gonadotropin-releasing hormone neurons. Endocr.Rev. 19(3), 302-330 (1998).

[27] d'Anglemontde Tassigny X, Fagg LA, Carlton MB et al. Kisspeptin can stimulate gonadotropin-releasing hormone (GnRH) release by a direct action at GnRH nerve terminals. Endocrinology 149(8), 3926-3932 (2008).

** Study which highlights the possible direct effect of Kisspeptin on GnRH nerve terminals within the hypothalamic median eminence.

[28] Rometo AM, Krajewski SJ, Voytko ML et al. Hypertrophy and Increased Kisspeptin Gene Expression in the Hypothalamic Infundibular Nucleus of Postmenopausal Women and Ovariectomized Monkeys. J Clin.Endocrinol.Metab (2007).

[29] Smith JT, Clay CM, Caraty A et al. KiSS-1 messenger ribonucleic acid expression in the hypothalamus of the ewe is regulated by sex steroids and season. Endocrinology 148(3), 1150-1157 (2007).

[30] Smith JT, Popa SM, Clifton DK et al. Kiss1 neurons in the forebrain as central processors for generating the preovulatory luteinizing hormone surge. J.Neurosci. 26(25), 6687-6694 (2006).

** Study which demonstrates activation of Kisspeptin neurons within the hypothalamic AVPV (but not Arc) during the rodent LH surge.

[31] Smith JT, Dungan HM, Stoll EA et al. Differential regulation of KiSS-1 mRNA expression by sex steroids in the brain of the male mouse. Endocrinology 146(7), 2976-2984 (2005).

** Demonstration of the dual regulation of hypothalamic Kisspeptin neurons by sex steroids, in rodents.

[32] Herbison AE. Estrogen positive feedback to gonadotropin-releasing hormone (GnRH) neurons in the rodent: the case for the rostral periventricular area of the third ventricle (RP3V). Brain Res.Rev. 57(2), 277-287 (2008).

[33] Witkin JW, Xiao E, Popilskis S et al. FOS expression in the gonadotropin-releasing hormone (GnRH) neuron does not increase during the ovarian steroid-induced GnRH surge in the rhesus monkey. Endocrinology 135(3), 956-961 (1994).

[34] Jayasena CN, Chaudhri OB, Nijher GK et al. Kisspeptin potently increases reproductive hromone release in women with hypothalamic amenorrhoea: a potential novel therapy for infertility. Presented at: Society for Endocrinology BES 2009 Meeting. Harrogate, UK, 16-19 March 2009.

[35] Horikoshi Y, Matsumoto H, Takatsu Y et al. Dramatic elevation of plasma metastin concentrations in human pregnancy: metastin as a novel placenta-derived hormone in humans. J.Clin.Endocrinol.Metab 88(2), 914-919 (2003).