Evidence for two distinct KiSS genes in non-placental vertebrates that encode kisspeptins with different gonadotropin-releasing activities in fish and mammals

TL;DR: The data are the first to provide conclusive evidence for the existence of a second KiSS gene, KiSS-2, in non-placental vertebrates, whose product is likely to play a dominant stimulatory role in the regulation of the gonadotropic axis at least in teleosts.

About: This article is published in Molecular and Cellular Endocrinology.The article was published on 2009-11-27 and is currently open access. It has received 224 citations till now. The article focuses on the topics: Kisspeptins & Sea bass.

Summary

2.1. Animals and tissue sample collection

• Sea bass (sbs) and medaka (md) (Carbio strain) were obtained from stocks at IATS, whereas zebrafish (zf) were obtained from a local pet shop.
• Brain and gonads were collected from male and female prepubertal and adult sea bass (n = 1-4 for each sex and developmental stage) in January, February, March and April.
• Besides, different somatic tissues were also collected from the samples in January.
• All the in vivo experiments were conducted in accordance with the European Union normative for use of experimental animals.

2.2. Molecular cloning of two KiSS-like genes in the sea bass

• To amplify fragments of sea bass KiSS-1, degenerate PCR primers were designed based on 12 conserved amino acids from the medaka, zebrafish, Fugu and Tetraodon putative KiSS-1 that included the kisspeptin-10 sequence ((Y/F)NLN(S/P)FGLR(Y/F)GK-NH 2 ).
• Secondary and nested PCRs were performed by using a primer (AP2) complementary to the adaptor sequence provided with the kit and degenerate and nested KiSS-1 PCR primers (second PCR sense, K2: 5´-AACYCITTYGGYCTCCGYTWYGGNAA-3´; antisense K4: 5´-CGGAGRCCRAANGRRTTIAGRTTRWA-3´).
• Then, specific primers corresponding to the 5' and 3' ends of KiSS-1 (sense kiss1-15 5'-GATGCCACGACTCATTGTCGCTC-3'; antisense kiss1-16 5'-AATCTTTAACCAACATTTTTATTGAG-3') and KiSS-2 (sense kiss2-16 5'-GGCACGAGGAGACACACACAC-3'; antisense kiss2-17 5'-TAA CCATACAGTGCTTTTATTGTG-3').
• CDNAs were used to amplify the full-length on a sea bass brain cDNA by using a proofreading DNA polymerase (Pfu DNA polymerase; Stratagene).

2.3. Sequence database searches, alignments, phylogenetic and genome mapping analyses

• All TBLASTN searches against the genome assemblies of different vertebrate species and retrieval of data for synteny analysis were performed at Ensembl (Ensembl Genome Browser, http://www.ensembl.org).
• The sequence of the sea bass KiSS-1 and KiSS-2 kisspeptin-10 motifs were used to query the putative gene sequences in other vertebrates.
• Multiple amino acid sequence alignments were generated with ClustalX (1.83) (Thompson et al., 1997) .

2.4. RNA isolation and RT-PCR analysis

• Total RNA was extracted from fish tissues with TRI-REAGENT according to the manufactures´ instructions.
• For expression analysis, two sets of gene-specific primers located in two different exons were designed for sea bass, medaka and zebrafish KiSS-1 and KiSS-2 genes (Table 1 ).
• RT-PCR analyses were performed using a touchdown PCR approach.
• The elongation factor-1 (ef1) gene was used in the three fish species as reference gene using specific primers (Table 1 ).

2.6. Administration of kisspeptins to sea bass

• In a first experiment, the ability of sbsKiSS-1 and sbsKiSS-2 peptides to elicit luteinizing hormone (LH) and follicle-stimulating hormone (FSH) secretion was assessed in prepubertal fish (1-year-old sea bass; 100 g in body weight, BW) by intramuscular (i.m.) administration in vivo at a dose of 250 ng of each peptide per gram of BW.
• In both experiments animals injected phosphate buffered saline served as controls.
• Blood samples were collected by caudal puncture, and plasma was separated by centrifugation at 2.500xg for 30 min at 4ºC and stored at -20ºC.

2.7. Administration of kisspeptins to rats

• To allow delivery of kisspeptins into the lateral cerebral ventricle, the animals were implanted, under ether anesthesia, with i.c.v. cannulae lowered to a depth of 4 mm beneath the surface of the skull; the insert point was 1 mm posterior and 1.2 mm lateral to bregma, as described elsewhere (Navarro et al., 2004a; 2005a; b) .
• Animals injected with physiological saline (0.9% NaCl) served as controls.
• Animals injected with physiological saline (0.9% NaCl) served as controls.

2.8. Hormonal analyses

• For hormonal tests in fish, plasma LH levels were determined using a validated enzyme-linked immunoassay for sea bass (Mateos et al., 2006) .
• Sea bass FSHR activation was measured by changes in luciferase activity (relative light units, RLU).
• Intra-and interassay coefficients of variation were respectively 6.9% and 9.5%.
• In rat experiments, serum LH and FSH levels were determined in a volume of 25-50 l using a double-antibody method and radioimmunoassay kits supplied by the NIH (Dr. AF Parlow, NIDDK National Hormone and Peptide Program; Torrance, CA, USA).

3.1. Cloning of sea bass KiSS-1 and KiSS-2

• Using degenerate primers and the Universal GenomeWalker Kit, the authors obtained two PCR fragments of 110 bp and 190 bp that corresponded to genomic sequence 5' upstream from kisspeptin-10 conserved motif of the sea bass KiSS-1 and KiSS-2 genes, respectively, and a 200 bp fragment containing KiSS-2 sequence 3' downstream from kisspeptin-10.
• The complete sea bass KiSS-1 cDNA has a length of 561 bp.
• The first 20 amino acids in KiSS-1 and the first 15 amino acids in KiSS-2 were predicted to constitute the putative signal peptide.
• Each of these genes contains two coding exons and one intron located in a similar position of the coding region.

3.2. Identification, phylogenetic and synteny analyses of KiSS-1 and KiSS-2 genes in vertebrates

• Searches in different available genome databases evidenced that KiSS-1 and KiSS-2 are also present in other vertebrate species such as the sea lamprey, zebrafish, medaka, Xenopus, and the non-placental mammal platypus, while some of the screened genomes showed only one of these two genes.
• Besides, comparison of the chromosomal regions containing KiSS-1 or KiSS-2 in different species uncovered a conserved synteny within each gene (Fig. 2B-C ).
• Although medaka and stickleback show a highly syntenic region between them.
• On the other hand, in all the fish species analyzed KiSS-2 was followed by the same genes in one side (LDHB, SLC25A3, TMPO and STRAP), whereas GYS2, C12orf39 and GOLT1B mapped on the other side except for medaka where some variation was found.
• This is the case of the pairs GOLT1A/GOLT1B, PLEKHA5/PLEKHA6, PIK3C2B/PIK3CG or ETNK1/ETNK2.

3.3. Tissue distribution of KiSS-1 and KiSS-2

• Results showed that sbsKiSS-1 and sbsKiSS-2 were predominantly expressed in brain and gonadal tissues (testis and ovary).
• In addition, sbsKiSS-1 was clearly expressed in the heart in juvenile males (Fig. 3A -upper left panel).
• In medaka, mdKiSS-1 was expressed in brain and testis, with lower levels of expression in eye and skin of adult males and in the ovary, gill and eye in adult females (Fig. 4 -upper panel).
• A faint expression was found in spleen, adipose tissue, liver and intestine of adult females.
• On the other hand, tissue distribution of zfKiSS-2 was limited to brain and ovary with low levels of expression in the testis (Fig. 4 -lower panel).

3.4. Temporal mRNA expression of KiSS-1 and KiSS-2 during the reproductive season of sea bass

• Considering the notable levels of expression of sbsKiSS-1 and sbsKiSS-2 in the brain, testis and ovary of adult and juvenile sea bass from both sexes at the beginning of the reproductive season , sample collection was monthly extended throughout the whole reproductive season (February-April).
• Particularly, in juvenile females sbsKiSS-1 expression in the brain was weaker than that of sbsKiSS-2, which showed robust expression levels during the whole reproductive period (Fig. 3B -lower left panel).
• In gonadal tissues, while the level of expression of sbsKiSS-1 was weak in the testis and ovary of adult males and females from January to April, notable expression levels were observed in the testis of juvenile males for the same period, whereas in juvenile females, expression of sbsKiSS-1 in the ovary was restricted to January.
• The authors also detected moderate levels of expression of sbsKiSS-2 in the gonads of adult males and females.
• In juvenile fish, modest expression levels of sbsKiSS-2 were observed in the testis, whereas in the ovary high levels of expression were detected in January, which declined in February, and became undetectable for that point onwards (Fig. 3B -upper right panel).

3.5. Effects of administration of kisspeptins on LH and FSH secretion in sea bass

• Functional analyses were conducted to test the gonadotropin-releasing capacity of sbsKiSS-1 and sbsKiSS-2 peptides in their homologous species.
• Systemic (i.m.) administration of both kisspeptins to prepubertal sea bass evoked significant elevations in circulating LH levels at 120 min after injection (Fig. 5A ).
• While sbsKiSS-2 elicited a 4-fold increase in mean LH concentrations over the corresponding preinjection levels, sbsKiSS-1 peptide induced only a 2-fold increase.
• In the same vein, maximal FSH secretory response of 2-fold increase was observed at 60 min after sbsKiSS-2 peptide injection, while sbsKiSS-1 peptide was able to induce only a moderate not significant increase of FSH secretion at 120 min after injection (Fig. 5B ).
• In addition, testing of the ability of both peptides to elicit LH secretion in vivo in 2-yr-old adult male sea bass revealed that only sbsKiSS-2 peptide elicited an increase of LH levels at 120 min after injection, which due to sample variability fell shortly below statistical significance (Fig. 5C ).

3.6. Effects of administration of kisspeptins on LH and FSH secretion in rats

• Functional analyses also included testing of the effects of sea bass kisspeptins in a heterologous mammalian species, the rat.
• Considering that sbsKiSS-1 is identical to rat Kp-10, and in order to avoid supra-physiological stimulation, sub-maximal, equimolar doses of kisspeptins were selected for central (i.c.v.; 100 pmol/rat) and systemic (i.p.; 15 g/rat) administration, in keeping with previous studies on the gonadotropin releasing effects of rat Kp-10.
• Finally, systemic (i.p.) administration of neither sbsKiSS-1 nor sbsKiSS-2 peptides induced further increases in serum FSH levels, at any time-point studied (Fig. 6D ).

4. Discussion

• This is the first description of the co-existence of two kisspeptin coding genes, namely KiSS-1 and KiSS-2, within the same species, the teleost fish sea bass.
• In addition, the functional tests reported herein evidence that, despite full conservation of KiSS-1 decapeptide from fish to mammals, the KiSS-2 peptide is likely provided with a much potent gonadotropin releasing activity in sea bass, thus suggesting a dominant role of this non-mammalian kisspeptin in the control of the gonadotropic axis, at least in teleosts.
• In a third scenario, both paralogs lose a different subset of functions, complementing each other to fulfill the ancestral one (sub-functionalization).
• In conclusion, the authors provide herein the first evidence for the co-existence of two KiSS genes, namely KiSS-1 and KiSS-2, in the genome of non-placental vertebrates.
• Negative control for PCR which was performed using sterile water as template, also known as C -.

Figures

Figure 2. Phylogenetic analysis and genomic organization of vertebrate KiSS-1 and KiSS2 genes. (A) Neighbor-joining bootstrap consensus tree. Bootstrap values (%) from 10000 replicates are indicated for each tree node. An alignment of amino acid sequences corresponding to exon 2 was used to generate the tree. Ambiguously aligned sequences and gaps were removed before phylogenetic analysis. (B) Synteny of KiSS-1 containing regions in vertebrate genomes. In mouse and rat the proximity between KiSS-1 and GOLT1A (number 7) indicates that the sequence of KiSS-1 was found within GOLT1A. Those genes boxed in human Chr1 are located in other chromosomes in that species. (C) Synteny of KiSS-2 containing regions in vertebrate genomes. Those genes boxed in mouse and rat are located in the indicated chromosome. In both schematic representations, not to scale, numbers on the boxes (and colour coding) indicate different genes, whose names are shown above. Orthologous genes are represented as a square, and those cases where paralogs were found in KiSS-1 and KiSS-2 analyzed regions are represented as an oval form. A black bar between genes indicates that these are not contiguous. Dashed boxes for KiSS-1or KiSS-2 represent absence of the gene. Numbers in brackets on the right indicate which chromosome (Ch), scaffold (S) or linkage group (LG) the cluster is found on. The gene searches were done at Ensembl against the genome assemblies of human (Hs) (Homo sapiens) (assembly NCBI 36), mouse (Mm) (Mus musculus), rat (Rn) (Rattus norvegicus) (assembly RGSC 3.4), opossum (Md) (Monodelphis domestica) (assembly MonDom 5), platypus (Ornithorhynchus anatinus) (assembly Ornithorhynchus_anatinus-5.0), chicken (Gg) (Gallus gallus) (assembly WASHUC2), lizard (Anolis carolinensis) (assembly AnoCar1.0), Xenopus (Xenopus tropicalis) (assembly Version 4.1), medaka (Ol) (Oryzias latipes) (assembly Zv7), zebrafish (Dr) (Danio rerio) (assembly Zv7), Fugu (Fr) (Fugu rubripes) (Ensembl 49), Tetraodon (Tn) (Tetraodon nigroviridis) (assembly Version 8), stickleback (Ga) (Gasterosteus aculeatus) (assembly BROAD S1) and sea lamprey (Petromyzon marinus)

Table 1. Gene-specific primers1 used for RT-PCR analysis

Figure 6. Analysis of the effects of central (i.c.v.; 100 pmol/rat) and systemic (i.p.; 15 g/rat) administration of sbsKiSS-1 and sbsKiSS-2 peptides on LH (A-B) and FSH (C-D) secretion over a 120-min period in adult male rats. Hormonal values are the mean ± SEM of 10 independent determinations. (*) P < 0.05.

Frequently asked questions

Q1. What are the contributions mentioned in the paper "Evidence for two distinct kiss genes in non-placental vertebrates that encode kisspeptins with different gonadotropin-releasing activities in fish and mammals" ?

Roa et al. this paper showed that kisspeptins, a family of structurally related peptides encoded by the KiSS-1 gene with ability to activate the G-protein coupled receptor 54 ( GPR54 ), play a major role in the regulation of the gonadotropic axis, with essential functions in the timing of puberty onset and the control of gonadotropin secretion. 

Q2. What are the future works mentioned in the paper "Evidence for two distinct kiss genes in non-placental vertebrates that encode kisspeptins with different gonadotropin-releasing activities in fish and mammals" ?

In any case, further research is needed to understand which driving forces led to these different fates in vertebrates. These data add further strength to the pivotal role of kisspeptins in the control of vertebrate reproduction, and illustrate the complex evolutionary process of this neuroendocrine system. 

Q3. What amino acids were predicted to constitute the putative signal peptide?

The first 20 amino acids in KiSS-1 and the first 15 amino acids in KiSS-2 were predicted to constitute the putative signal peptide. 

Q4. What is the effect of sbsKiSS-2 on the circulating LH?

while sbsKiSS-2 elicited a 4-fold increase in mean LH concentrations over the corresponding preinjection levels, sbsKiSS-1 peptide induced only a 2-fold increase. 

Q5. How many amino acids are in the sea bass KiSS-1 and KiSS-2 cDNA?

Using degenerate primers and the Universal GenomeWalker Kit, the authors obtained two PCR fragments of 110 bp and 190 bp that corresponded to genomic sequence 5’ upstream from kisspeptin-10 conserved motif of the sea bass KiSS-1 and KiSS-2 genes, respectively, and a 200 bp fragment containing KiSS-2 sequence 3’ downstream from kisspeptin-10. 

Q6. What was the PCR reaction used to amplify the full-length cDNA?

cDNAs were used to amplify the full-length on a sea bass brain cDNA by using a proofreading DNA polymerase (Pfu DNA polymerase; Stratagene). 

Q7. What was the PCR reaction used to obtain the cDNA ends of the sea bass?

PCR reactions using gene-specific primers in combination with primers annealing in the UNI Zap-XR vector were performed to obtain the cDNA ends. 

Q8. What was the luciferase activity measured on the plates?

After 6 h incubation luciferase activity was quantified directly on the plates using the Steady-Glo Luciferase Assay System (Promega) and the ULTRA Evolution (TECAN) detection platform. 

Q9. What is the effect of sbsKiSS-2 on sea bass?

In addition, testing of the ability of both peptides to elicit LH secretion in vivo in 2-yr-old adult male sea bass revealed that only sbsKiSS-2 peptide elicited an increase of LH levels at 120 min after injection, which due to sample variability fell shortly below statistical significance (Fig. 5C). 

Q10. What was the effect of sbsKiSS-2 on sea bass?

Systemic (i.m.) administration of both kisspeptins to prepubertal sea bass evoked significant elevations in circulating LH levels at 120 min after injection (Fig. 5A).