The Kisspeptin System in Male Reproduction

The kisspeptin system includes the cleavage products Kiss1 precursor and kisspeptin receptor (Kiss1R). It was originally discovered and studied in cancer metastasis, but the identification of KISS1/KISS1R gene mutations causing hypogonadotropic hypogonadism (HH) revealed unexpected effects in reproduction. Nowadays, the kisspeptin system is the main central gatekeeper of the reproductive axis at puberty and adulthood, but it also has a widespread functional role in the control of endocrine functions. At the periphery, Kiss1 and Kiss1R are expressed in the testes, but the need for kisspeptin signaling for spermatogenesis and sperm quality is still unclear and debated. This brief manuscript summarizes the main findings on kisspeptin and male reproduction; upcoming data on sperm maturation are also discussed.


Introduction
In the last 25 years, the discovery of kisspeptins and their receptor Kiss1R strongly contributed to the discovery of new signaling mechanisms in biological systems [1]. The Kiss1 gene was first discovered in 1996 as an anti-metastasis gene in melanoma cell lines [2]. This gene encodes a precursor that is then cleaved into shorter peptides (Kp-54 or metastin, Kp-14, Kp-13, Kp-10), all sharing the carboxy-terminal region and capable of activating the G coupled membrane receptor Kiss1R; the 10-aminoacid-long Kp-10 represents the shorter peptide for Kiss1R activation [3,4].
Since the Kiss1 discovery, the knowledge of the kisspeptin system in cancer significantly enlarged, nowadays revealing anti-metastasis, cancer-promoting or pro-metastasis activity, depending on cancer types, cancer microenvironment and steroid receptor status in the cancer cells [5,6]. However, the discovery in 2003 that inactivating mutations in the human KISS1R gene [7,8] caused hypogonadotropic hypogonadism (HH) with deficiencies in gonadotropin-releasing hormone (GnRH) secretion, gonadotropin discharge and infertility opened new perspectives in the biological action of the kisspeptin system, revealing unexpected roles in the central control of reproduction.
Furthermore, the characterization of the system in non-mammalian vertebrates [9] and the expression of Kiss1 and Kiss1R (at both mRNA and protein levels) in peripheral tissues, such as gonads, placenta, pancreas, adipose tissues, liver or vasa, additionally enlarged the biological role of the system from cancer progression to reproduction, fertility and energy homeostasis, suggesting that the system has more general roles in the control of endocrine functions in vertebrates [1].
In this Communication, the main findings on kisspeptin activity in the control of male reproduction are briefly summarized, with a focus on studies published during the last 5 years on the effects of kisspeptin at the periphery. Upcoming data from epididymal spermatozoa (SPZ) and future perspectives are also discussed.

Kiss1 and Kiss1R in the Central Control of Reproduction
The production of gametes is under the control of the hypothalamus-pituitarygonadal (HPG) axis and requires endocrine, paracrine and autocrine communications among the hypothalamus, which drives the pulsatile secretion of the GnRH; the pituitary, which is responsible for the discharge of gonadotropins (Follicle Stimulating Hormone (FSH) and Luteinizing Hormone (LH)); and gonads, which, in turn, produce sex steroids [10].
Notably, the gain of function and the loss of function mutations in KISS1/KISS1R genes cause precocious puberty and HH, respectively, in both human and animal models [7,8,[11][12][13], as a consequence of the precocious stimulation or impairment of the HPG axis at the hypothalamic level. In fact, hypothalamic GnRH-secreting neurons possess on their membranes Kiss1R [14], and express and secrete the neurohormone GnRH under the control of kisspeptin, which is produced in the brain by neuronal populations located within the arcuate nucleus and the anteroventral periventricular nucleus [14].
The molecular mechanism that mediates kisspeptin action on GnRH gene expression involves kisspeptin-induced dynamic chromatin modifications [15,16]. A kisspeptin-response element (KsRE) between −3446 and −2806 bp of the mouse GnRH gene enhancer has been detected in GnRH-neuronal cell lines and in transgenic mice. KsRE represents a binding site for the transcription factor Orthodenticle Homeobox 2 (Otx-2). Kisspeptin treatment causes nucleosome-depleted DNA in KsRE, induces the transcription of the Otx-2 gene and the synthesis of the Otx-2 protein, promotes Otx-2 protein binding to KsRE, and consequently activates GnRH gene transcription [15]. Specifically, kisspeptin significantly increased histone 3 (H3) acetylation at lysine (K) 14 and K27 and the trimethylation of H3K4 within the KsRE, but did not affect the dimethylation of H3K9, thus resulting in being effective on well-known markers of active chromatin, but devoid of action on gene repression markers. Furthermore, a chromosome conformation capture assay revealed that kisspeptin promoted the functional formation of a chromatin loop between KsRE and the downstream located neuron-specific element to gain the transcriptional activation of the GnRH gene [16] (Figure 1).
Kisspeptin neurons are also intermediate in the sex-steroid mediated feedback mechanisms on reproduction [14] and convey on the HPG axis several environmental cues, thus modulating reproduction with epigenetic mechanisms [17,18]. In this respect, diet or exposure to environmental toxicants centrally affect reproduction, modulating the activity of Kiss-secreting neurons. The best example in the field is the epigenetic modulation of puberty, a process that requires specific methylation changes in the Kiss1 and Kiss1R gene promoters [19] and the activity of the NAD + dependent deacetylase Sirtuin 1 (Sirt1) that epigenetically modulates Kiss1 expression within the hypothalamus [20]. In fact, reproduction strongly depends on energy availability and Sirt1 acts as metabolic sensors capable of activating or repressing gene expression through mechanisms involving the deacetylation of histone proteins, transcriptional factors or cofactors [21].

Kiss1 and Kiss1R in Testis and Spermatozoa
Spermatogenesis is a complex process modulated step-by step by the autocrine, paracrine and endocrine route. It needs the coordination between germ cell proliferation and death, meiotic division and differentiation events, and the key contribution of Leydig cells in the interstitium to produce sex-steroids, and Sertoli cells in the seminiferous tubules to provide structural and nourishment support to developing germ cells [10]. The kisspeptin system has been characterized in the testis of mammalian and non-mammalian vertebrates, revealing possible roles in the autocrine and paracrine intra-testicular communications, steroid biosynthesis, spermatogenesis progression and sperm functions, but also species-specific differences in localization and possible functions in turns [22][23][24].
Kisspeptin, but not GnRH, has been detected in human plasma and measured in different health conditions [12]. In males, circulating kisspeptin levels change in relation to fertility status, being significantly higher in fertile than in infertile men [25]. Some HH patients have high levels of kisspeptin in the plasma, but, after GnRH replacement therapy, circulating kisspeptin levels decrease as a consequence of the restored sex-steroid feedback mechanisms at hypothalamic levels [26]. However, gonadotropin stimulation is not always able to rescue testosterone biosynthesis and spermatogenesis in clinical cases of KISS1R inactivating mutations [27,28], suggesting the need for testicular Kiss1R signaling for steroidogenesis. Similarly, the specific reactivation of the Kiss1R gene in the GnRH secreting neuron of KISS1R −/− knockout mice does not restore spermatogenesis, further confirming the need of the intratesticular kisspeptin signal for successful spermatogenesis [29]. By contrast, testosterone replacement in KISS −/− mice that exhibit HH restores plasma and intratesticular testosterone levels and sustains spermatogenesis until the production of spermatozoa capable of fertilizing eggs in vitro, but treated mice failed to impregnate females [30].
The administration of kisspeptin usually promotes spermatogenesis in intact animal models [31][32][33]. Nevertheless, the kisspeptin-dependent over-stimulation of the HPG axis causes testis damage if prolonged [34] and the switch-off of the HPG axis through Kiss1R desensitization [35].
Post-natal testis development and Leydig cells maturation require kisspeptin signaling, and synergistic effects involving both the hypothalamic and LH-dependent intratesticular production of kisspeptin have been suggested in rodents [36]. In fact, centrally produced kisspeptin activates the HPG axis, leading to hypothalamic GnRH secretion and gonadotropin LH production by the anterior pituitary; in turn, LH signaling in Leydig cells induces Kiss1 expression through cyclic adenosine monophosphate and the activation of the protein kinase A pathway [37]. In vitro effects on GnRH expression in Leydig cells [38] and the modulation of intratesticular GnRH system, estradiol and testosterone levels in non-mammalian vertebrates [32,39] have also been reported. Recently, the co-culture of germ and somatic cells revealed kisspeptin-dependent effects on spermatogenesis progression [40], confirming observations in more physiological conditions, such as ex vivo testes explants [31,32,39].
Hence, in spite of in vivo, ex vivo and in vitro observation revealing a possible role in Leydig cells physiology and steroid-secreting activity, or spermatogenesis progression as well [24], the intratesticular role of the kisspeptin system is far from being elucidated and still debated [23].
Additionally, humans, bulls, rodents and frogs express Kiss1R [24] and the system has been characterized in goat, hamster, mouse and rat epididymis [24,41]. Nevertheless, the need for kisspeptin to gain SPZ competence for fertilization has been poorly investigated and remains unclear. The localization of Kiss1R differs in humans and mice, being mainly localized in the post-equatorial segment in humans [42] and acrosomic region in mice [43]. Currently, the modulation of the kisspeptin system in SPZ by specific agonists/antagonists in physiological conditions has been reported in human [42] and mouse spermatozoa [43] only. These studies revealed Kp-13-dependent effects on human sperm hypermotility [42] and effects of Kiss1R antagonism by Kp-234 on the fertilizing ability of mouse SPZ collected in the epidydimal tail [43]. Recently, we characterized the kisspeptin system in dog and rat SPZ [41,44], providing evidence of Kiss1R trafficking in SPZ head during the transit from caput to cauda epididymis (Figure 1). In the dog, a model close to humans, the combined use of flow cytometry, epifluorescence microscopy and Western blot on specific membrane protein fractions revealed Kiss1R on membrane-intact SPZ collected from the epididymal tail. In particular, the presence of Kiss1R on SPZ surface parallels the acquisition of the typical features for successful epidydimal maturation (i.e. protamination rate and motility) [44]. In the rat, immunofluorescence analysis carried out on permeabilized SPZ collected in caput and tail epididymis revealed that Kiss1R shifts from the posterior region of the SPZ head in the epididymal caput to the perforatorium in the epidydimal tail [41]. Interestingly, by dot blot assay, Kiss1 was detected in the epididymal fluid of both rats and dog [41,44] and, in rat, a specific ELISA assay was used to measure significant Kiss1 levels in the epididymal fluid and in the plasma, used as a positive control [41]. Thus, Kiss1R trafficking seems to be a marker of appropriate sperm maturation, and kisspeptin signaling may represent a signal for SPZ storage within the epididymis. The main limitations of these studies are the lack of functional data on the activity of Kiss1R in SPZ collected at different tracts of the epididymis in both physiological and pathological conditions. Thus, further studies are required in the field. Interestingly, kisspeptin levels have been measured in seminal plasma and blood plasma in a large cohort of healthy men, revealing higher kisspeptin levels in the seminal plasma than blood and a positive association between sperm quality and kisspeptin within the seminal plasma [45]. Hence, it could be interesting to analyze the kisspeptin system in SPZ-defective animal models, but also in the seminal plasma of normospermic, subfertile and infertile men as an additional sperm quality parameter.

Conclusions
In conclusion, kisspeptin signaling has a fundamental role in the central control of reproduction at puberty and adulthood, but additional studies are required to elucidate the need for peripheral kisspeptin signaling in spermatogenesis and sperm functions. Nevertheless, upcoming data revealed the differential localization of Kiss1R in the SPZ head during epididymal maturation. In parallel, the detection of significant Kiss1 amounts in seminal plasma and epididymal fluid strongly encourages additional studies in the field and candidates for the kisspeptin system to become a promising marker and target in evaluating and addressing male fertility troubles in the near future.

Conflicts of Interest:
The author declares no conflict of interest.