118 Identification of KISS1 in the Domestic Cat

Abstract

In the last decade, a substantial dataset has been acquired to demonstrate the critical role of kisspeptins, a family of neuropeptides derived from the KISS1 gene, in the reproduction system of mammalian and non-mammalian species. The KISS1 gene initial product can be cleaved into a 54-amino acid protein, and further degrade from the N-terminus into shorter but still active peptides (Kp-10, Kp-13, and Kp-14). The resulting peptides share a common C-terminal RF-amidated motif that leads to a strong binding with kisspeptin receptor. The Kp-10 peptide is well conserved between studied species and is thought to be essential and sufficient for the activation of kisspeptin receptor signalling pathways. In several species, the sequence of KISS1 has been obtained, and synthesised Kp-10 and Kp-54 have been successfully administered to induce ovulation. However, no studies have yet been performed to identify the KISS1 gene and its products in the domestic cat. Therefore, the aim of our work was to clone Felis catus KISS1 (fKISS1) and obtain its full sequence. Total RNA was isolated from the hypothalamus of a pubertal domestic cat collected postmortem and its quality was ensured using Agilent Bioanalyzer 2100 (RNA integrity number 8.2). Initial primers were designed based on the sequence deduced from the comparison of predicted domestic cat mRNA KISS1 sequence (XM_003999477.2) and published dog, human, cow, pig, goat, mouse, and rat mRNA KISS1 sequences. The partial mRNA fKISS1 sequence obtained here was used to design gene-specific primers for 5′ RACE and 3′ RACE PCR (GeneRacer Kit, Invitrogen, Carlsbad, CA, USA). For sequencing, 6 colonies were used per 5′ end and 3′ end of cloned fKISS1 cDNA. Additionally, the presence of KISS1 mRNA was checked via PCR in the ovaries, collected after ovariohysterectomy from domestic cats during inactive, follicular, and luteal ovarian stages. Nucleotide sequencing revealed that fKISS1 cDNA is 723 bp, and the open reading frame consists of 450 bp encoding a 149-amino acid polypeptide (kisspeptin precursor). Comparison of the overall amino acid sequence of the kisspeptin precursor revealed that domestic cat kisspeptin precursor exhibits similarity of 84, 78, 75, 68, 64, 56, and 52% to those of Hawaiian monk seal, pig, naked mole rat, human, mouse, musk shrew, and dog, respectively. The core sequence of kisspeptin, Kp-10, was highly conserved compared with other mammalian and non-mammalian species (100%, Hawaiian monk seal; 100%, mouse; 100%, pig; 100%, African clawed frog; 100%, naked mole rat; 100%, musk shrew; 90%, human; 90%, dog). Apart from hypothalamic tissue, fKISS1 has been identified in the ovaries on all stages. Obtained here results could be used for development of ovulation induction protocols in the endangered feline species. Moreover, a curious high similarity of kisspeptin sequence reported here to other induced ovulators may contribute to the research on ovulation mechanism in the domestic cat.