Recently, we described three Kissr genes in the genome of a basal teleost, the European eel, providing the first evidence of the existence of three Kissr genes in a teleost species (Pasquier et al., 2012). Furthermore, we described four Kissr in the genome of a non-teleost actinopterygian, the spotted gar, as well as in the genome of a basal sarcopterygian, the coelacanth (Pasquier et al., 2012). This provided the first evidence for four Kissr genes in vertebrate species and revealed a larger diversity of Kissr than previously described.

So far, no Kissr sequence has been reported in chondrichthyans. Our search in the elephant shark genome has only led to the identification of multiple partial sequences, corresponding at least to two Kissr (unpublished data). Ongoing sequencing of other chondrichthyan genomes, such as dogfish (Scyliorhinus canicula), may provide more insights into the Kissr diversity in the sister group of osteichthyans.

To further assess the Kiss diversity in vertebrates, we re-investigated the presence of these genes in the genome of the elephant shark, the coelacanth, the spotted gar, and the European eel, representative species from four groups of relevant phylogenetical positions. Most of the vertebrate Kiss genes are made of two exons except for some mammalian Kiss1, including human Kiss1, pig (Sus scrofa) Kiss1, and mouse Kiss1, that are made of three exons (Figure 1). However, the CDS of all the Kiss described so far are split on two exons (Figure 1). In fact the first of those two exons encodes the signal peptide while the final exon encodes mainly the mature peptides including the conserved Kp(10) (Figures 1 and 4; Tomikawa et al., 2010, 2012; Cartwright and Williams, 2012; Tena-Sempere et al., 2012). Considering that the Kiss gene sequences are highly variable among species except for the sequence encoding the Kp(10), we focused our prediction on the ORF containing this sequence. We performed TBLASTN in the four genomes, resulting in the identification of several ORF containing conserved sequences encoding for Kp(10).

Two Kiss genes in the European eel genome. The two ORFs containing the sequences encoding Kp(10) are 296 and 327 bp long, respectively (Figure A1 in Appendix). Once translated, each of them leads to a peptidic sequence encompassing a putative Kp(10): YNWNSFGLRY [European eel Kp1(10)] and FNRNPFGLRF [European eel Kp2(10)], respectively (Figure 4). The C-terminal end of the Kp1(10) sequence is followed by a GK-Stop motif, while the Kp2(10) sequence is followed by a GKR motif (Figure A1 in Appendix). The sequences “X-G-Basic-Basic” or “X-G-Basic” are characteristic of the conserved proteolytic cleavage and alpha-amidation sites of neuropeptides (Eipper et al., 1992).

Two Kiss genes in the spotted gar genome. The two ORFs containing the sequences encoding Kp(10) are 348 and 300 bp long, respectively (Figure A2 in Appendix). Once translated, each of them leads to a peptidic sequence presenting a putative Kp(10): YNWNSFGLRY [spotted gar Kp1(10)] and FNFNPFGLRF [spotted gar Kp2(10)], respectively (Figure 4). The C-terminal ends of these two sequences are followed by a GKR motif (Figure A2 in Appendix).

Three Kiss genes in the coelacanth genome. The three ORFs containing the sequences encoding Kp(10) are 363, 396, and 81 bp long, respectively (Figure A3 in Appendix). Once translated, each of them leads to a peptidic sequence encompassing a putative Kp(10): YNWNTFGLRY [coelacanth Kp1(10)], FNFNPFGLRF [coelacanth Kp2(10)], and FNWNSFGLRF [coelacanth Kp3(10)], respectively (Figure 4). The C-terminal ends of the Kp1(10) and the Kp2(10) sequences are followed by a GKR motif, while the Kp3(10) is followed by a GKK motif (Figure A3 in Appendix). Seven amino-acids up-stream the sequence of the Kp3(10), a stop codon appears (Figure A3 in Appendix) suggesting that coelacanth Kiss3 gene could have a different intro-exon structure compared to what has been described so far or it can suggest that this gene is no longer expressed.

A third Kiss gene in the elephant shark genome. While two Kiss (Kiss1 and Kiss2) were previously identified in the elephant shark genome (Lee et al., 2009), we were able to localize a new ORF of 315 bp containing a third sequence encoding a Kp(10) (Figure A4 in Appendix). Once translated, it leads to a peptidic sequence encompassing a putative Kp(10): YNLNSFGLKF [elephant shark Kp3(10)] (Figure 4). The C-terminal end of this peptide is followed by a GKR motif (Figure A4 in Appendix).

The alignment of 56 kisspeptin precursors revealed a high variability of their amino-acid sequences except for the sequences corresponding to Kp(10) and its few surrounding amino-acids which, in contrast, are highly conserved (data not shown). Such a pattern, which is representative of many other neuropeptide precursors, provides poor phylogenetic information within alignment matrix. This lack of information makes the use of phylogenetic analysis inappropriate to establish homology relationships within this kind of peptide precursor family.

Among the new Kiss genes predicted in the present study, four of them encode novel Kp(10) (Figure 4). The singularity of the elephant shark Kp3(10) is the presence of a lysine (K) instead of an arginine (R) at the ninth position. The coelacanth Kp1(10) provides the first case of a threonine (T) at the fifth position. The coelacanth Kp3(10) is the only one to present both a phenylalanine (F) at the first position and a serine (S) at the fifth position. The European eel Kp2(10) presents at its third position an arginine (R), which possesses different physical and chemical properties from amino-acids commonly present at this position. Up to now, only the musk shrew (Suncus murinus) Kp1(10) presented an arginine at the third position and it was demonstrated that its kisspeptin system was involved in the reproductive function as in other mammals (Inoue et al., 2011). Since the impacted positions by the amino-acid substitutions have not been characterized as highly critical for Kp(10) functional properties (Gutiérrez-Pascual et al., 2009; Curtis et al., 2010), those novel Kp(10) may have conserved their functionality. Since Kp(10) is considered as the smallest required sequence to specifically bind to the receptor (Kotani et al., 2001), it could be of interest to test all those peptides in future pharmacological studies in order to assess their structure/function relationships.

In order to classify the different Kiss paralogs, we performed a syntenic analysis of the Kiss neighboring genes. We considered the following vertebrate representatives: mammals (human), birds (chicken), squamates (lizard), amphibians (Xenopus), basal sarcopterygian (coelacanth), non-teleost actinopterygians (spotted gar), and teleosts (zebrafish, stickleback, and European eel). Our syntenic analysis demonstrated that the Kiss genes are localized in three different genomic regions.

The human Kiss1, Xenopus Kiss1a, coelacanth Kiss1, spotted gar Kiss1, and zebrafish Kiss1 are positioned in genomic regions containing common loci, including TEAD3, NAV1, PPP1R12B, PPFIA4, MYBPH, KCNC4, REN, GOLT1A, PLEKHA6, PPP1R15B, PIK3C2B, and SYT6, thus exhibiting well conserved synteny (Figure 5A). This supports the orthology of these Kiss genes, all considered as Kiss1 genes. Syntenic analysis suggests that the stickleback, lizard, and chicken genomes do not contain any Kiss1 gene, although the above-mentioned neighboring genes are present in the corresponding genomic regions (Figure 5A). The peptidic sequence of eel Kiss1 presents many similarities to the other Kiss1, but the eel Kiss1 gene is located on too small scaffolds to contain any other gene, preventing from any syntenic analysis.

The lizard Kiss2, coelacanth Kiss2, spotted gar Kiss2, zebrafish Kiss2, stickleback Kiss2, and European eel Kiss2 genes are positioned in genomic regions containing common loci including STRAP, PLEKHA5, GOLT1B, C12orf39, GYS2, LDHB, KCNJ8, ABCC9, CMAS, SYT10, NAV3, PPFIA2, and KCNC2, thus exhibiting well conserved synteny (Figure 5B). This supports the orthology of these Kiss genes, all considered as Kiss2 genes. Syntenic analysis suggests that human and chicken genomes do not contain any Kiss2 gene, although the above-mentioned neighboring genes are present in the corresponding genomic region (Figure 5B).

The coelacanth Kiss3 and the Xenopus Kiss1b genes are positioned in genomic regions containing common loci, including TEAD2, PIH1D1, and ALDH16A1, thus exhibiting well conserved synteny (Figure 5C). This supports the orthology of these two Kiss genes, both considered here as Kiss3 genes. Syntenic analysis suggests that human, lizard, spotted gar, and teleost genomes do not contain any Kiss3 gene, although the above-mentioned neighboring genes are present in the corresponding genomic regions (Figure 5C). Syntenic analysis also suggests that the whole considered region is absent from the chicken genome.

The syntenic analysis also allowed us to investigate the origin of the multiple Kiss genes found in vertebrates. The three conserved genomic regions, presenting Kiss genes, also comprise other paralogs from 11 gene families: TEAD (4 paralogs), NAV (3 paralogs), PPFIA (4 paralogs), KCNC (4 paralogs), GOLT1 (2 paralogs), PLEKHA (4 paralogs), PPP1R15 (2 paralogs), PIK3C2 (3 paralogs), SYT (4 paralogs), GYS (2 paralogs), and PTH (2 paralogs) (Figures 5A–C). The members of those families are present among the three Kiss syntenic regions and they also delineate a fourth conserved region (Figure 5D), which does not present any Kiss gene in the osteichthyan representative species studied so far. The four conserved regions delineated by the 11 gene families can be considered as paralogous (tetra-paralogon).

Considering the reconstruction of the ancestral vertebrate chromosomes, their linkage to the tetra-paralogons in the human genome (Nakatani et al., 2007) and our localization of the four Kiss syntenic regions in the human genome (on Chromosomes 1, 11, 12, and 19), we can hypothesize that the Kiss tetra-paralogons resulted from the duplications of one single genomic region localized on the proto-chromosome-D of the vertebrate ancestor. Therefore, we can infer that the current three Kiss genes may have resulted from a single ancestral gene duplicated through 1R and 2R that occurred in early steps of vertebrate evolution (Figure 6).

Multiple loss events in sarcopterygians and actinopterygians. The 1R and 2R events should have resulted in four different Kiss genes in vertebrates. Since the fourth Kiss gene (referred to as Kiss4 in this study) has not been observed in any species studied so far, we can hypothesize an early loss of this gene after the 2R. As only a chondrichthyan, the elephant shark, and two sarcopterygians, the coelacanth and Xenopus, still present three Kiss genes, whereas all other species possess less than three Kiss, we can hypothesize multiple additional events of Kiss losses in vertebrates (Figure 3B).

Among the sarcopterygian lineage, in tetrapods, Kiss3 would have been lost in amniotes. Further alternative losses may have occurred in this lineage, with only Kiss1 remaining in eutherian mammals and only Kiss2 in squamates (lizard). Finally, additional losses would have led to the complete absence of Kiss in birds (Figure 3B). Among the actinopterygian lineage, an early loss of Kiss3 would have occurred since it is lacking in the actinopterygian species investigated so far (Figure 3B).

No impact of the teleost-specific 3R on Kiss number in current species. In the actinopterygian lineage, the teleost-specific 3R and the presence of two Kiss in a non-teleost actinopterygian, the spotted gar, implied the potential existence of at least four Kiss genes in the early teleost history. However, our study showed that so far the largest number of Kiss exhibited by current teleosts, including the eel, is two. Furthermore, each teleost Kiss is orthologous to a different tetrapod Kiss, indicating that no teleost-specific Kiss exists. Synteny analysis revealed that each of the four Kiss genomic regions present in the spotted gar is duplicated in teleosts in agreement with the 3R event but that duplicated Kiss genes are lacking (Figures 5 and 6). This suggests an early loss of duplicated Kiss genes suppressing the impact of the 3R on the number of Kiss in teleosts (Figure 3B). Additional deletion may have led to the presence of only Kiss2 in gasterosteiforms (the stickleback; Figure 3B). Kiss evolutionary history was punctuated by numerous loss events through vertebrate radiation (Figure 3B).

In the current gnathostomes, we observed a maximum of four Kissr but only three Kiss paralogs. This difference suggests that this lineage inherited the four Kissr copies resulting from the 1R and 2R, whereas the fourth Kiss may have been lost before or at an early stage of the gnathostome emergence (Figure 3). This situation was observed in an early sarcopterygian, the coelacanth, while an even larger difference in Kissr (four) and Kiss (two) numbers was found in the spotted gar, reflecting an additional independent loss of Kiss in the actinopterygian lineage. An un-matching number of Kissr (three) and Kiss (two) was also observed in an early teleost, the European eel, while additional losses may have led to equal numbers of Kissr and Kiss in more recent teleosts (two or one according to the species). These variations in Kissr/Kiss numbers reflect different timing of Kiss and Kissr loss events. Those hypotheses suggest that Kiss losses occurred independently among the different gnathostome lineages and also independently from the Kissr losses.

The hypothesis of independent Kiss and Kissr evolutionary histories is also strengthened by the comparison of the gene sets present in species with even matching numbers of Kiss and Kissr. For example, lizard, and stickleback both present the Kiss2 gene, whereas they possess different Kissr, i.e., Kissr-4 in the lizard and Kissr-2 in the stickleback (Figure 3). The same observation can be done comparing the kisspeptin systems of platypus and zebrafish, which both present the Kiss1 and Kiss2 genes, whereas their sets of receptors are completely different, i.e., Kissr-1 and Kissr-4 in platypus versus Kissr-2 and Kissr-3 in zebrafish (Figure 3). These observations strongly suggest a large variety of Kiss and Kissr combinations, resulting from independent loss events. These data shed new lights on the evolution of the kisspeptin system in vertebrates and challenge the former hypothesis of a conservation of Kiss/Kissr couples across vertebrate evolution. This diversity among vertebrates opens new research avenues for comparative physiology and endocrinology of kisspeptin system.

A few in vitro studies using recombinant receptors have showed cross-reactivities between various kisspeptins and kisspeptin receptors (Biran et al., 2008;Lee et al., 2009; Li et al., 2009). For instance, Lee et al. (2009) tested the specificity of recombinant human GPR54 (Kissr-1 according to our nomenclature), zebrafish GPR54-1, and -2 (Kissr-3 and Kissr-2 according to our nomenclature), and Xenopus GPR54-1a, -1b, and -2 (Kissr-1, Kissr-4, and Kissr-2 according to our nomenclature) toward various kisspeptins. They showed that human, zebrafish, and Xenopus kisspeptins were able to activate all the receptors with differential intra and inter-specific ligand selectivity. Such cross-reactivity could have promoted the independence of the Kiss and Kissr evolutionary histories. This could explain the situation of species presenting un-matching numbers of Kiss and Kissr genes, as well as the high variability of Kiss/Kissr gene combinations across vertebrate species. Another study, using goldfish recombinant GPR54a and GPR54b (Kissr-3 and Kissr-2 according to our nomenclature), revealed that the ligand potency strikingly differed depending on the responsive element used in the reporter gene construction (Li et al., 2009). These data showed the difficulty to define specific Kiss/Kissr couples based only on pharmacological properties.

In the case of the presence of multiple Kiss/Kissr genes in a given species, anatomical relationships between projections of Kiss neurons and target cells expressing Kissr may provide further cues for determining Kiss/Kissr functional couples. Thus, in zebrafish which possess two Kiss genes and two Kissr genes, in situ hybridization and immunocytochemical studies localized the Kiss1 neurons in different nuclei from Kiss2 neurons (Servili et al., 2011). Moreover, Kiss1 neurons are projecting to Kiss1r (Kissr-3 according to our nomenclature) expressing cells, while Kiss2 neurons are projecting to Kiss2r (Kissr-2 according to our nomenclature) expressing cells (Servili et al., 2011). This reveals anatomically separated kisspeptin systems and distinct specific Kiss/Kissr functional couples in zebrafish (Servili et al., 2011). In striped bass, another teleost possessing two Kiss genes and two Kissr genes, in situ hybridization and laser capture microscopy coupled to quantitative PCR showed, in contrast, that Kiss1 and Kiss2 were co-expressed in neurons of the hypothalamus, indicating promiscuous Kiss synthesis sites (Zmora et al., 2012). However, the two Kissr of the striped bass were expressed in different brain cells, indicating that the kisspeptin systems are not fully redundant (Zmora et al., 2012). All these data underline the importance of investigating the gene diversity, the anatomical organization and the functional properties of the kisspeptin system in various species, regarding the potential high variability of this system among vertebrates.