Kisspeptins and their Affiliated Receptors' Effects on the Regulation of the Female Mammalian HPG-axis, with Associated Gene Mutations
Contents
Introduction
In 1996, Lee et al. identified kisspeptins as a product of the KiSS1 gene, a metastasis suppressor gene, in malignant melanomas (Pratheesh, et al., 2011). The term kisspeptins (Kp's) is comprehensively used to communally describe a family of neuropeptides that have an Arg-Phe-NH2 pattern at the C-terminus, which are primarily expressed in distinct neuronal populations of the hypothalamus (Pratheesh, et al., 2011; Roa, et al., 2011).
Kp's act as mediators of various physiological and hormonal signals in both male and female mammalian and non-mammalian vertebrates (D'Anglemont de Tassigny & Colledge, 2010). They play an essential role as regulators of crucial aspects of development and function of the reproductive axis, also termed the hypothalamic-pituitary-gonadal axis (HPG-axis) (Roa, et al., 2011). The importance of Kp and its associated receptor; KiSS1r, formally known as G-protein receptor – 54 (GPR54), was demonstrated in 2003 following mutations of the peptide or receptor (Pineda, et al., 2010). These mutations manifest phenotypically as precocious puberty (Sonigo & Binart, 2012) or delayed, perhaps absent puberty; hypogonadotropichypogonadism (De Roux, et al., 2003; Seminara, et al., 2003; Roseweir & Miller, 2009).
As Kp is the natural ligand for the KiSS1r (Lee, et al., 1999), mechanisms of Kps' actions as a molecular switches for puberty and reproductive development are a current source of research. Kp's stimulate the secretion of gonadotropins from the pituitary through exciting the release of Gondatropin Releasing Hormone (GnRH) from the forebrain after the activation of KiSS1r by GnRH neurons (Pratheesh, et al., 2011).
Kisspeptins and the HPG-Axis
The HPG-axis is a complex system governing a number of bodily functions, but is mainly responsible for development, reproduction and ageing in vertebrates (Klein, 2012). This system regulates reproduction in the body by pulsatile GnRH secretion. This crucial neurotransmitter of the HPG-axis induces a corresponding pulsatile release of gonadotropins, namely luteinizing hormone (LH) and follicle stimulating hormone (FSH), from the anterior pituitary gland (Roa, et al., 2011; Oakley, et al., 2010). Both of these gonadotropins are of prime importance in female reproductive physiology, initiating ovarian follicle development, and ovulation (Hanache, et al., 2012; Klein, et al., 2012; Oakley, et al., 2010).
The KiSS1 gene encodes a 145 amino acid (aa) long propeptide from which a 54aa sequence, known as Kp-54, is cleaved (West, et al., 1998). It is believed that Furin, a prohormone convertase enzyme, is responsible for cleaving Kp54 from its 145aa long precursor (Kotani, et al.,2001). Shorter sequences have also been discovered such as kisspeptin-10, -13, -14, which all have the same Agr-Phe-NH2 pattern at the C-terminus (Oakley, et al., 2009; West, et al., 1998).
The relevance of these short peptides is not known, but all kisspeptins show equal affinity for and efficiency at their corresponding receptor (Kotani, et al., 2001; Muir, et al., 2001; Ohtaki, et al., 2001). It has thus been concluded that the C-terminal of the peptide is responsible for the binding and activation at the receptor (Kotani, et al., 2001).
GnRH neurons are located mainly in the preoptic area of the hypothalamus, which, when activated, releases GnRH. GnRH travels via the hypophyseal portal system to the pituitary gland, where LH and FSH are secreted (D'Anglemont de Tassigny & Colledge, 2010). These two hormones are regulated by a negative feedback loop of sex steroids, oestrogen and progesterone, produced by the gonads (Klein, 2012; Roa, et al., 2011; Oakley, et al., 2010).
According to D'Anglemont de Tassigny & Colledge in 2010, the KiSS1 gene is predominantly expressed in the hypothalamus, more specifically in two main populations; one in the arcuate nucleus (ARC) and one in the anteroventral periventricular nucleus (AVPV). They further describe that the distribution also shows interspecies differences and, moreover, disparity between sexes. A larger number of cells expressing the gene are found in the AVPV of females (Clarkson, et al., 2009). This is most likely linked to the organizing effect of sex steroids during neonatal sexual maturation of the brain (Kauffman, et al., 2007). Most GnRH neurons in the hypothalamus express KiSS1r with interspecies differences. Using double immunofluorescence, a staining method to observe several antigens in one sample, it was discovered that 85% of GnRH neurons in the medial eminence (ME) of rat’s brains express KiSS1r (Hatsai, et al., 2009; Irwig, et al., 2009).
The firing rate of GnRH neurons can be directly depolarized by Kp which increases the GnRH secretion (Liu, et al., 2008). Zhang, et al., (2009) and Piclelca Fortuna et al., (2008) have found evidence suggesting Kp's are also able to regulate GnRH secretion through intermediary neurons. It is commonly concluded that Kp's effect the LH and FSH secretion indirectly via the GnRH pathway. However, some studies have given us reason to believe that in addition to working on a hypothalamic level, Kp can act directly on the gonadotropes in the pituitary gland; stimulating LH and FSH secretion directly. In 2007, Kauffman et al., mentioned the expression of KiSS1r in the pituitary gland, but discarded the idea of Kp having a direct effect on a pituitary level. They concluded that GnRH secretion caused by Kp was a result of direct activation of GnRH neurons. Three years later, Oakley, et al., (2010) revisited the possibility for Kp activation directly through the gonadotropes in the pituitary gland. This was based on the findings of functional KiSS1r in the pituitary, in addition to detecting Kp in ovine hypophyseal portal blood. Nevertheless, there is still ongoing controversy about this dual activation, as GnRH secretion via direct Kp stimuli is proven to be relatively low and the Kp content of ovine hypophyseal portal blood is not elevated during the preovulatory LH surge (Smith, et al., 2008; Oakley, et al., 2010). Shortly after the administration of exogenous Kp a significant increase in plasma LH and FSH levels were noted. This surge has proven to be sufficient to induce estrous cycles in prepubescent animals and to cause ovulation. In 2009 Sébert, et al., conducted a study on Kp's effect on LH secretion and ovulation in anestrous ewes. Of the treated animals, 75% ovulated after 24hours of a 43hour infusion of Kp. Similar increases in LH/FSH secretion was proved by Pineda, et al. (2010) when Kp was intra-cerebrally administrated to rats.
The majority of research of Kp effects on the HPG-axis has focused on LH secretion, but recently efforts to chart Kp's role in FSH secretion have greatly increased. Experiments on rats demonstrated that a dosage of Kp 100 times higher than LH is necessary to secrete FSH (Navarro, et al., 2005). Serbert, et al., (2012) found that ewes infused with exogenous Kp showed an increase in GnRH secretion by elevated levels of FSH and LH in the plasma. This resulted in a preovulatory LH surge approximately 22hours into the infusion. After the surge, plasma levels of gonadotropins decreased. FSH concentration dropped back to basal level, whilst LH concentration was still significantly elevated. They concluded that the drop in gonadotropins was caused by the negative feedback loop of estrogen. Studies also show that prolonged exogenous Kp administration can lead to a down-regulation of the GnRH receptors and in turn LH and FSH secretion, due to desensitization of the KiSS1r (Oakley, et al., 2010; Mead, et al., 2007). The pulse frequency of GnRH secretion has an impact on which gonadotropin hormone will be released. In 2012, Miller, et al., stated that "low GnRH pulse frequency is shown to favor FSH secretion, whilst high GnRH pulse frequency favors LH secretion".
Androgen (AR) and estrogen receptors (ERα) are not exhibited in GnRH neurons, thus it has long been suspected that another component is involved in regulating GnRH secretion via the negative/positive feedback system of sex steroids (Herbison & Theodesis, 1992; Huang & Harland, 1993). Kp neurons express both AR and ERα, thus their intimate contact with GnRH neurons has a crucial role in relaying feedback information (Clarkson, et al., 2008). Furthermore, Kp is believed to be linked to the suppression of GnRH/LH pulse during lactation, due to lower KiSS1 gene expression in the ARC region and lower KiSS1 receptor expression in the AVPV region of the hypothalamus in lactating animals (Yamada, et al., 2007).
In addition, Kp has been found to directly communicate with neurons containing nNOS (neuronal Nitric Oxide Synthase). Moreover, Nitric Oxide release is essential for the preovulatory GnRH surge, which is also mediated by Kp. Thus, it is evident that Kp has an extremely important role within the HPG-axis (Hanchate, et al., 2012).
Mutations of the KiSS1 Gene
As previously mentioned, Kp has a pivotal role in many of the bodies functions and of particular interest to recent research, the reproductive (HPG) axis. Both, Mead, et al. (2007) and Navarro, et al. (2005) refer to Kp as the ‘molecular switch for puberty’. Thus, if Kiss1 or Kiss1r has mutated it will have serious repercussions for the individual. These mutations may be spontaneous or genetically targeted: missense (point mutations) deletions or insertions, (Roseweir & Millar, 2009; Kauffman, et al., 2007; Tena-Sempere, 2006). Often such mutations reveal themselves as disorders such as Idiopathic Hypogonadotrophic Hypogonadism (IHH) (decreased activity of the gonads), delayed puberty, hypothalamic secondary amenorrhea (cessation of menstruation) and even precocious puberty (early onset puberty), (Sonigo, & Binart, 2012; D’Anglemont de Tassigny, & Colledge, 2010; Oakley, et al., 2009). Furthermore, mutations may cause infertility, poor gonadal growth and impaired gametogenesis; which, in females, results in the disruption of the normal estrus cycle leading to ovulation issues and lack of Corpus Lutea development (D’Anglemont de Tassigny, & Colledge, 2010). Roseweir and Millar (2009) explain that the affected individuals have ‘small ovaries and uteri, delayed vaginal opening, no maturation of follicles, decreased sexual behaviour... and low gonadotropin levels’. Accordingly, these mutations can have varying severity from loss of function to gain of function, whereas some are so mild that they decrease GnRH levels but allow normal bodily functioning (Roa, et al., 2011; D’Anglemont de Tassigny & Colledge, 2010; Millar, et al., 2010; Oakley, et al., 2009; Tena-Sempere, 2006).
There are many polymorphisms that exist for Kp in the female; resulting in the phenotypes described previously. Mutations of KiSS1r gene, such as R331X and X399R, cause IHH that results in decreased GnRH production (Mead, et al., 2007). These mutations are the result of the stop codon having been ‘moved’, thus elongating the sequence. Further Kiss1r gene mutations resulting in IHH are missense mutations C223R, R297L and L102P. These mutations, however, affect the receptor in a different way, resulting in impaired GnRH signaling. Of the three, C223R has the most severe effects. There are two other mutations of the Kiss1r known to cause IHH: a 155 nucleotide deletion and L1485 which is a single nucleotide deletion. Less is known of the 155 nucleotide deletion but the deletions do have different effects. L1475 has a similar effect to C223R, R297L and L102P (Roseweir & Millar, 2009; Mead, et al., 2007).
Additionally there are two other gene mutations which cause IHH: TAC3 and TAC3R. Although these are not strictly Kp mutations they are thought to have a direct effect on Kp and thus GnRH secretion. These mutations are of the gene which encodes a protein called NKB and its receptors. NKB is believed to regulate the secretion of Kp along with another protein called Dyn (Pineda, et al., 2010).
There are two known mutations of the Kiss1r gene that cause precocious puberty: R386P and P110T (Roseweir & Millar, 2009). Oakley, et al. (2009) explains that these mutations cause ‘prolonged intracellular Kiss1r signalling in response to Kp’. Both IHH and precocious puberty caused by such mutations present other symptoms also; such as hypothalamic secondary amenorrhoea, (Roseweir & Millar, 2009).
Concluding Remarks
Kisspeptins regulate various aspects of the female reproductive pathways, many of which are not yet fully understood. It is a vigorously researched topic exploring the mechanisms and mutations of the Kp gene and its associated receptor, KiSS1r.
The maintenance of hormonal equilibrium within the HPG-axis, is under both direct and indirect influence of Kp's. Gondatropin releasing hormone from the hypothalamus is under direct duress from Kp, which, acting indirectly through GnRH, induces the release of both LH and FSH (Roa, et al., 2011; Oakley, et al., 2010). It is evident, from conclusive research, that interference in this mechanism, via insertion, deletion or point mutations, can dramatically alter the physiological reproductive development and function of an individual. Additional research, linked to the successful functioning of Kp, is predicted on the proteins NKB and Dyn. These proteins are thought to work as “auto-regulators of Kp output onto GnRH neurons” (Pineda, et al., 2010). With a more thorough understanding not just of the effects of Kp on the HPG-axis, but also the factors that control and regulate Kp expression in an individual, possible therapeutic applications of this gene may be comprehended.
Surmounting the basic understanding, mapping and function of Kp, researchers believe that the regulation of reproductive cycles can thus be manipulated. For example, earlier estrus cycles in domestic mammals could have a potentially constructive impact on agricultural economy (Caraty, et al., 2012). Studies were conducted with the aim of inducing well-timed and coordinated ovulations in exogenously Kp-treated animals. An LH surge was noted in all treated animals, with an understanding that Kp had the capability to be used to activate the reproductive function of animals that are acyclic (Caraty, et al., 2012).
Furthermore, it is thought that this exogenous administration of Kp could potentially be a therapeutic aid to treat “infertility, premature and delayed puberty, and prostatic or metastatic cancers” (D’Anglemont de Tassigny & Colledge, 2010). The prospective therapeutic and biological applications of the study of Kp's regulatory pathways in both veterinary and human medicine are undeniable.
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